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The Journal of Neuroscience, September 15, 1999, 19(18):7804-7811
Fast Excitatory Nicotinic Transmission in the Chick Lateral
Spiriform Nucleus
Yi
Nong,
Eva M.
Sorenson, and
Vincent A.
Chiappinelli
Department of Pharmacology, The George Washington University
Medical Center, Washington, DC 20037
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ABSTRACT |
The lateral spiriform nucleus (SpL) in the chick mesencephalon
contains functional nicotinic receptors and receives a cholinergic fiber projection. We now use double-label immunohistochemistry to
demonstrate that choline acetyltransferase-immunopositive fibers in the SpL and in the cholinergic fiber tract lateral to the nucleus are associated with fibers expressing the  5 and/or 3 nicotinic receptor subunits as determined by mAb35 immunoreactivity. This morphological evidence suggests that there might be synapses between the cholinergic fibers and the dendrites of SpL neurons. Whole-cell recordings from SpL neurons in current-clamp mode revealed EPSPs evoked by stimulation of the cholinergic fiber tract lateral to the
SpL. These EPSPs increased in amplitude in the presence of bicuculline.
Further addition of the nicotinic antagonist dihydro- -erythroidine (DHE) to the buffer significantly attenuated them. Almost all of the
remaining EPSP was blocked by 6,7-dinitroquinoxaline-2,3-dione. In the
presence of an antagonist cocktail that isolated the nicotinic responses, a fast, monosynaptic nicotinic EPSP or EPSC was
evoked. In some neurons, the nicotinic EPSP resulted in the generation of an action potential. The nicotinic nature of the evoked
response was confirmed by blockade of the EPSPs or EPSCs with nicotinic antagonists, including DHE, D-tubocurare, and
mecamylamine. The nicotinic response was insensitive to low
concentrations (10-100 nM) of methyllycaconitine,
indicating that typical 7-containing receptors were not involved.
The results demonstrate that endogenously released acetylcholine
generates EPSPs that can elicit action potentials by acting at
postsynaptic nicotinic receptors on SpL neurons.
Key words:
nicotinic; evoked; neurotransmission; lateral spiriform
nucleus; cholinergic; 5 nicotinic receptor subunit
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INTRODUCTION |
Several types of central neurons are
known to express postsynaptic nicotinic receptors as evidenced by
recording of nicotinic potentials or currents in response to exogenous
nicotinic agonists and blockade of these responses by nicotinic
antagonists. However, there are few reports of fast synaptic nicotinic
neurotransmission in brain (Role and Berg, 1996 ). We have previously
chosen the chick lateral spiriform nucleus (SpL) as a likely nucleus to
contain nicotinic cholinergic synapses based on the findings that the SpL receives a cholinergic projection and that SpL neuronal cell bodies
and processes have among the highest densities of
3H-nicotine binding sites in the brain
(Sorenson et al., 1989; Sorenson and Chiappinelli, 1992). Intracellular
recordings with sharp electrodes demonstrated that activation of
nicotinic receptors with exogenous agonists caused SpL neurons to
depolarize and generate action potentials (Sorenson and Chiappinelli,
1990). Further studies using whole-cell patch-clamp recordings have
found that the nicotinic receptors on SpL neurons are likely to consist
of more than one combination of nicotinic receptor subunits because
they have differential sensitivities to trimethaphan and show four
different sizes of single-channel currents (Weaver et al., 1994; Weaver
and Chiappinelli, 1996). Nicotinic agonists were found to have
presynaptic, as well as postsynaptic, effects because they increased
the frequency of spontaneous GABAergic IPSCs recorded from SpL neurons
(McMahon et al., 1994).
A study by Ullian and Sargent (1995) returned the focus of our
attention to the cholinergic afferents to the SpL. These authors suggested that most of the nicotinic receptors on SpL neurons are not
localized at synapses on the somata of SpL neurons as shown by
double-label immunofluorescence for individual nicotinic receptor
subunits and a synaptic vesicle protein. We wanted to take their study
one step further and determine whether the cholinergic afferents in the
SpL were associated with structures containing nicotinic receptors. We
have begun by examining the double labeling of the SpL with mAb35 for
the 3/5 nicotinic receptor subunits and antiserum against choline
acetyltransferase (ChAT) (the synthetic enzyme for acetylcholine). We
now report that ChAT-positive afferents are associated with
mAb35-immunopositive fibers, some of which appear to be processes of
SpL neurons. To further test the hypothesis that cholinergic afferents
synapse onto the dendrites of SpL neurons, we have stimulated the
cholinergic fiber tract lateral to the SpL while performing whole-cell
patch-clamp recording from SpL neurons. The electrophysiological
results establish that SpL neurons receive fast synaptic
neurotransmission mediated by nicotinic receptors.
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MATERIALS AND METHODS |
Immunohistochemistry. Three newly hatched White
Leghorn chicks (Truslow Farms, Chestertown, MD) were decapitated, and
their brains were removed. The mesencephalon was dissected out and
submersion-fixed in 1% paraformaldehyde in 0.1 M
phosphate buffer for 4 hr at 4°C. The brains were then put in either
30% sucrose for cryoprotection or 0.1 M PBS, pH
7.35, until sectioning. Transverse sections containing the SpL
were cut on a cryostat (50 µm) or on a vibratome (100 µm). The
sections were double or single immunolabeled for ChAT and/or the
5/3 nicotinic receptor subunits. Specifically, free-floating sections were washed and then incubated with one or both of the primary
antibodies for 24-72 hr. The primary antiserum for choline acetyltransferase, a rabbit antiserum against the chicken enzyme, was
the generous gift of Miles Epstein (University of Wisconsin) (Johnson
and Epstein, 1986). It is well characterized and has been previously
used in our laboratory (Sorenson et al., 1989). mAb35 is a rat
monoclonal antibody that recognizes the 1, 3, and 5 nicotinic
receptor subunits (Research Biochemicals, Natick, MA) (Tzartos et al.,
1981). It also is well characterized and has been used for
immunolabeling nicotinic receptors in the chick brain, particularly in
the SpL (Swanson et al., 1983; Ullian and Sargent, 1995). The dilution
for the ChAT antiserum was 1:2500 or 1:5000, and for mAb35 it was
1:6600. The incubation buffer consisted of 0.1 M
PBS containing 5% normal goat serum and 0.3% Triton X-100. The
sections were given three washes in PBS and then incubated overnight in
buffer containing both secondary antibodies. The secondary antibodies
were produced in goat. The anti-rabbit antibody was conjugated to Cy3
and the anti-rat antibody was conjugated to Cy5 (Jackson
ImmunoResearch, West Grove, PA). After washing, the sections were
mounted on slides and coverslipped using 90% glycerol, 10% PBS, and
4% propyl gallate as the mounting medium. Coverslips were sealed with
fingernail polish and stored in the dark at 4°C until imaging.
Specificity was determined by omitting either one or both of the
primary antibodies from the incubations or by omitting the secondary antibodies.
Imaging was done on a Bio-Rad (Hercules, CA) MRC-1000 confocal
microscope equipped with a krypton-argon laser. Cy3 was excited with
the 568 nm excitation line of the laser, and emissions were collected
using emission filter 605/32. Cy5 was excited with the 647 nm line of
the laser, and emission was collected using the 680/32 filter. The
laser was attenuated with a 3, 10, or, occasionally, 30% transmission
neutral density filter. Images were collected with either a 20 or 60×
planapo objective with numerical apertures of 0.7 and 1.4, respectively. Image collection was done with signal averaging. Images
were collected sequentially for the two different fluorophores in
double-labeled sections. Scan speed, low-signal, and iris settings
depended on the particular specimen under observation.
Electrophysiology. SpL slices (400 µm) were prepared from
chick embryos at 18 d of incubation. Brain slices were cut with a
vibrating tissue slicer in cold, oxygenated buffer. The composition of
the external recording buffer was (in mM): 126 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.2 Na2HPO4, 25 NaHCO3 and 10 glucose bubbled with 95%
O2-5% CO2. The slice was
continuously superfused at 4 ml/min at room temperature. Drugs were
applied by bath perfusion.
The SpL neurons were visualized with an Axioskop microscope (Zeiss,
Jena, Germany) for whole-cell patch-clamp recording. Patch pipettes
were made by a two-stage microelectrode puller and had a resistance of
4-7 M after filling with an internal solution containing (in
mM): 140 K-gluconate, 10 HEPES, 5.0 EGTA, 5 Mg-ATP, and 2 MgCl2. QX314 (5 mM) was added to the
pipette solution to block Na+ transients
when only voltage-clamp experiments were planned. Conventional
patch-clamp techniques were used with an Axopatch-200B amplifier (Axon
Instruments, Foster City, CA) in either the voltage- or current-clamp
configurations. In voltage clamp, holding potential was between  60
and 70 mV. The series resistance
(Rs) was calculated by
adjusting the cell capacitance and Rs
potentiometers on the amplifier. The
Rs was 9.4 ± 0.9 M
(n = 25). The Rs
compensation was set at 75-90% (lag, 10 µsec). Cholinergic
afferents were stimulated at 0.05 Hz at an intensity between 100 and
900 µA and a duration of 100-900 µsec with a concentric bipolar
electrode placed at the lateral side of the SpL. The distance between
the bipolar and recording electrodes ranged from 0.2 to 1 mm. The EPSC
or EPSP was recorded with a video cassette recorder (Vetter,
Rebersburg, PA). The data were sampled by pClamp 6 software (Axon
Instruments) through a Digidata 1200 DMA interface.
Analysis of evoked synaptic events was performed by Clampfit 6.03 software (Axon Instruments) and SigmaPlot 4.0 (SPSS Inc., Chicago, IL).
Seven consecutive traces of evoked responses were averaged. The peak
current amplitude was calculated by averaging three adjacent points at
the peak. The decay time constants were determined using a simplex
algorithm by fitting EPSCs with the exponential function
I = A * exp(t/ ) + C, where A is the amplitude coefficient, and is the decay time constant. In some cases, the sum of two exponential
functions was used. Statistics were performed with SigmaStat version
1.0 (SPSS Inc). The data were expressed as mean ± SEM.
Differences between means were examined by paired t test.
Drugs were obtained as follows: eserine, atropine sulfate,
()-nicotine bitartrate, 6,7-dinitroquinoxaline-2,3-dione (DNQX), and
bicuculline methiodide from Sigma (St. Louis, MO);
2-amino-5-phosphovalerate (AP-5), methyllycaconitine (MLA), and
mecamylamine from Research Biochemicals; and D-tubocurare
from Calbiochem (San Diego, CA). Dihydro--erythroidine (DHE) was
a gift from Merck, Sharp & Dohme Research laboratories (Rahway, NJ).
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RESULTS |
Colocalization of ChAT and mAb35
Single- or double-labeled sections treated with mAb35 contained
mAb35-positive fibers in the tract lateral to the SpL (Fig. 1A,C).
The cells in the SpL itself were heavily labeled with mAb35, but there
were no labeled cells present within the cholinergic fiber tract
lateral to the SpL. It was not possible to determine whether the
mAb35-positive fibers lateral to the SpL originated from SpL neurons.
Sections of the SpL single- or double-labeled for ChAT showed a
cholinergic fiber tract lateral to the SpL as reported previously (Fig.
1B,C). Within the nucleus, a lower
density of neuropil labeling was present, and no cells were
immunopositive. Double-labeled sections had a high density of overlap
of mAb35-positive fibers and ChAT-positive fibers in the tract lateral
to the SpL (Fig. 1C). Portions of the nucleus farthest from
the fiber tract appeared to have lower levels of ChAT fibers than the
rest of the nucleus. At higher magnification, it was apparent that
there was more interaction of ChAT-immunopositive fibers with
mAb35-positive fibers in the tract lateral to the SpL than in the SpL
itself (Fig. 1D-F).
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Figure 1.
Localization of ChAT and the 3/5 subunits in
the SpL. Sections containing the SpL were double labeled with mAb35 and
anti-ChAT antiserum. Sequential images of the labeling for each of the
antigens were collected and merged to demonstrate areas of
colocalization. mAb35 labeling is represented by red,
ChAT by green, and colocalization by
yellow in all images. A-C, At low
magnification, mAb35 is seen to label most SpL neurons and also fibers
lateral to the nucleus (A, arrowheads). A
cholinergic fiber tract is immediately lateral to the nucleus, and
cholinergic fibers emanate from the tract into the SpL
(B). C, A merge of images
A and B, demonstrates that mAb35-positive
fibers are localized in the cholinergic fiber tract. The highest
density of colocalization of the two antigens appears to be in the
cholinergic fiber tract. A-C were collected at normal
speed with the iris set at 4.9 mm. D-F, mAb35,
anti-ChAT, and a merge of the mAb35 and anti-ChAT immunolabeling,
respectively, in the same section. In the region in which the fiber
tract meets the SpL, there appear to be more cholinergic fibers
associated with mAb35-positive fibers within the fiber tract than with
SpL neurons. The arrowheads in F point
out mAb35-positive fibers dotted with ChAT immunoreactivity.
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Serial optical images through randomly selected neurons were obtained
consecutively for mAb35 and ChAT immunolabeling. When the individual
images were merged, it appeared that the ChAT labeling was
predominantly associated with mAb35-positive fibers rather than the
cell bodies (Fig.
2A-F). ChAT
immunolabeling would follow, or dot, mAb35-labeled fibers, but there
was little ChAT labeling at the surface of the cells. The ChAT- and
mAb35-labeled fibers were not identical (Fig.
2A-F). Furthermore, neurons in the nucleus semilunaris, the source of the cholinergic afferents in the SpL, were
immunopositive for ChAT but not for mAb35 (data not shown). Some
of the mAb35-immunopositive fibers that originate from SpL neurons may
be associated with ChAT fibers (Fig. 2C,D,
arrows). Control sections in which the primary antibodies or
secondary antibodies were omitted did not show any fluorescence.
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Figure 2.
Cholinergic afferents in the SpL are associated
with mAb35-immunopositive fibers. The figures are merged images of
mAb35 and ChAT labeling at 1 µm intervals through a neuron in the
SpL. Note that there do not appear to be ChAT-positive fibers
associated with the surface of the SpL neuron. Rather, the ChAT
labeling follows, or dots, the mAb35-positive fibers.
Arrowheads show examples of areas in which the two
antigens are in close proximity and may represent cholinergic synapses.
Arrows indicate processes of the neuron that may be
associated with ChAT fibers (C, D).
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Nicotinic receptors directly mediate fast
excitatory neurotransmission
Whole-cell recordings were performed in visually identified SpL
neurons (n = 127). Evoked monosynaptic responses were
elicited by electrical stimulation (0.05 Hz) lateral to the SpL
nucleus. EPSPs were elicited in 90% (36 of 40) of SpL neurons whose
resting membrane potentials were held at 60 mV in the current-clamp
mode. In some neurons, evoked EPSPs increased in amplitude with
increasing stimulation intensities. In six of these SpL neurons, the
EPSP reached threshold, and an action potential was generated (Fig. 3A, left). The
action potential threshold was 49.2 ± 0.6 mV (n = 6). The mean height of the evoked action potentials was 59.7 ± 2.9 mV, and the width at half the maximum of the action potential was
1.76 ± 0.08 msec. The height of the action potential was
determined by measuring from threshold to peak. DHE (10-60
µM), a nicotinic antagonist, blocked the evoked
spike but an EPSP remained (n = 3) (Fig. 3A,
right). In the presence of an antagonist cocktail to isolate
the nicotinic response, the nicotinic EPSP was large enough to generate
an action potential in some neurons (n = 3) (Fig.
3B, left). The action potential and underlying
EPSP could, under these conditions, be blocked by addition of DHE
(30 µM) to the superfusion buffer
(n = 3) (Fig. 3B, right). These
results indicate that activation of nicotinic receptors by synaptically released acetylcholine produces excitatory postsynaptic potentials that
can reach threshold and result in the generation of an action potential
in SpL neurons.
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Figure 3.
Excitatory synaptic transmission mediated by
nicotinic receptors. A, An action potential was
generated by electrical stimulation lateral to the SpL
(left). Stimulation was at 0.05 Hz (450 µA, 300 µsec). The nicotinic receptor antagonist DHE (30 µM)
blocked the action potential but did not completely block the EPSP
(right). B, In another SpL neuron, an
antagonist cocktail containing AP-5 (50 µM), DNQX (40 µM), bicuculline (10 µM), and atropine (1 µM) was given to isolate the nicotinic response. The
isolated nicotinic EPSP increased in amplitude with increasing
stimulation intensity and, at 700 µA, 500 µsec
(left), was sufficient to generate an action potential.
DHE (30 µM) completely blocked the action potential
and EPSP (right).
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In the presence of bicuculline (10 µM), a
GABAA antagonist, 43% of neurons tested (17 of
40) generated action potentials after stimulation of afferents (Fig.
4A, second
trace). DNQX (20-40 µM) blocked the
evoked action potentials, but an EPSP was still present (Fig.
4A, third trace). Nearly all of the
remaining EPSP was sensitive to DHE (60 µM)
(Fig. 4A, last trace). In the
voltage-clamp mode, both outward and inward currents were recorded when
the holding potential of the neurons was between 55 and 60 mV (Fig. 4B, first trace). Bicuculline (10 µM) blocked the outward current, indicating
that it is mediated by GABAA receptors (Fig.
4B, second trace). The blockade of the
outward current caused an increase in the amplitude of the inward
current. A portion of the inward current was sensitive to DNQX (40 µM) (Fig. 4B, third
trace), indicating that it was caused by activation of AMPA
receptors. Nearly all of the remaining inward EPSC could be blocked by
DHE (60 µM) (Fig. 4B,
last trace).
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Figure 4.
Evoked responses are mediated by nicotinic,
glutamatergic, and GABAergic receptors. A, In
current-clamp mode, electrical stimulation (450 µA, 300 µsec)
lateral to the SpL elicits an EPSP in an SpL neuron
(first trace). When GABAA
receptors are blocked with bicuculline, the identical stimulus produces
an action potential (second trace). Further addition of
DNQX blocks the action potential (third trace), and most
of the remaining EPSP is blocked by subsequent DHE application
(last trace). B, After a 55 min washout
of all the antagonists, the experiment was repeated in voltage-clamp
mode.
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Properties of the evoked nicotinic response
To isolate the nicotinic EPSCs, the slice was superfused with the
following antagonist cocktail (in µM): 50 AP-5, 40 DNQX, 10 bicuculline, and 1 atropine. Furthermore, in cells examined only in the voltage-clamp mode, QX314 (5 mM) was frequently
added to the internal pipette solution to block transient
Na+ currents. Under these conditions, the
sensitivity of the remaining EPSCs to a variety of nicotinic receptor
antagonists was tested to help characterize the nicotinic receptor
subtype(s) mediating synaptic transmission. DHE (30 µM) significantly (83 ± 5.4%) reduced the EPSC
amplitude (n = 6) (Fig.
5A). The blockade of nicotinic
EPSCs by DHE usually took 4-8 min, and complete recovery could be
achieved by washing out DHE for 20-30 min.
D-Tubocurare (30 µM)
blocked 85 ± 3.4% of the response amplitude in 3-5 min (n = 6) (Fig. 5B). The response recovered
40-60 min after washing out the D-tubocurare.
Mecamylamine (10-20 µM) blocked 62-79% of the EPSC or EPSP amplitude in 10-15 min (Fig. 5C,
n = 4). Mecamylamine was difficult to wash out, and
60-80 min were required for the response to recover completely after
washout of drug was begun.
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Figure 5.
Properties of evoked nicotinic synaptic currents
recorded from SpL neurons. The evoked responses shown in
A-E were examined in the presence of an antagonist
cocktail containing AP-5 (50 µM), DNQX (40 µM), bicuculline (10 µM), and atropine (1 µM) to isolate the nicotinic EPSCs. In
A-C, superimposed traces of the evoked synaptic
currents are shown for SpL neurons before
(Control), during (Drug), and
after (Washout) exposure to the indicated nicotinic
antagonists. Holding potentials were 70 mV, and traces are averages
of seven consecutive responses. In D, MLA (100 nM) did not inhibit the evoked nicotinic EPSCs
(left). In contrast, 30 µM MLA
significantly inhibited the evoked nicotinic EPSCs in the same neuron
(right). In E, an I-V
plot of the evoked nicotinic EPSCs was obtained in a SpL neuron. The
nicotinic currents reversed near 0 mV and showed strong rectification
at positive membrane potentials.
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MLA is a selective antagonist of nicotinic receptors containing the
7 subunit in a concentration range of 10-100 nM
(Alkondon and Albuquerque, 1993). At higher concentrations, MLA blocks
all neuronal nicotinic receptors (Yum et al., 1996). In SpL neurons, MLA at 10-100 nM did not significantly decrease
(p > 0.05; paired t test) the
nicotinic EPSC amplitude (n = 5) (Fig. 5D,
left). In contrast to low concentrations of MLA, 30 µM MLA did inhibit the nicotinic EPSCs by
54 ± 3.4% (n = 4) (Fig. 5D,
right). These results suggest that the nicotinic receptors
mediating synaptic transmission in SpL neurons do not contain 7 subunits.
The evoked nicotinic synaptic currents were detected after a delay of
1.0-3.2 msec after the stimulus artifact. The peak current amplitudes
ranged between 10 and 117 pA with little variation between responses of
the same neuron. The evoked nicotinic synaptic currents had a mean rise
time of 6.7 ± 0.4 msec (n = 20) measured as the
time required for the current to rise from 10 to 90% of the peak
value. The decay phase of the currents could be fit well by a
monoexponential function (r2 > 0.95). The decay time constant ranged from 15.6 to 62.4 msec. The
current-voltage relationship of the nicotinic EPSC is shown in Figure
5E. The current did not reverse itself but came to a plateau
at ~0 mV. The extrapolated reversal potential is 0.6 mV (n = 6).
The effects of eserine on evoked EPSCs in the SpL
Eserine is an inhibitor of acetylcholinesterase, the degradative
enzyme for acetylcholine. In the presence of 10 µM
eserine, the amplitude of the EPSCs was reduced and the decay phase
dramatically prolonged (n = 6). In the example shown in
Figure 6A, the maximum amplitude was reduced from 117 to 72 pA, and the decay time constant () was increased from 32.2 to 2898 msec. It took 60-80 min of washout to recover from the effects of eserine. At a lower
concentration of 3 µM, eserine had less of an
effect on the maximum amplitude but altered the decay rate to a
two-exponential process (n = 4). In the example shown
in Figure 6B, the maximum amplitude was 32.1 pA in
control versus 28.7 pA in eserine. The single exponential rate of decay
in control ( value of 56.0 msec) changed to a two-exponential process in eserine with 1 of 38.9 msec and
Amp1 of 13.4 pA (56.8%), and
2 of 909 msec and Amp2
of 10.2 pA (43.2%). After washing eserine out for 60 min, the decay
rate was again a single exponential, similar to control ( value of
62.1 msec).
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Figure 6.
Effect of the acetylcholinesterase inhibitor
eserine on evoked nicotinic EPSCs. Holding potential was 70 mV, and
an antagonist cocktail containing AP-5 (50 µM), DNQX (40 µM), bicuculline (10 µM), and atropine (1 µM) was present throughout. All traces are averages of
seven consecutive evoked responses. A, At 10 µM, eserine dramatically prolongs the decay phase of the
EPSC and decreases the maximum current amplitude. B, At
3 µM, eserine has less of an effect on the maximum
amplitude but alters the decay phase.
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DISCUSSION |
The present study establishes that direct excitatory nicotinic
transmission occurs in the mesencephalic SpL and characterizes the
anatomical and functional elements of this central nicotinic synapse.
Presynaptic cholinergic fibers arise from the nucleus semilunaris and
form a fiber tract lateral to the SpL (present study; Sorenson et al.,
1989). The cholinergic fiber density is highest in the fiber tract with
individual ChAT fibers leaving the tract and entering the nucleus.
Tract tracing studies using anterograde transport of tritiated amino
acids have demonstrated that the fiber tract lateral to the SpL
originates in the nucleus semilunaris (Reiner et al., 1982). However,
based on the silver grain pattern they observed, Reiner et al. (1982)
suggested that nucleus semilunaris innervated the area immediately
lateral to the SpL and had only a slight projection to the SpL. The
merged images of double-labeled sections in the present study also
suggest that the cholinergic innervation of the SpL is not uniform. The areas of the SpL nearest the cholinergic tract appear to have more
cholinergic fibers than the areas of the SpL farthest away from the tract.
The mAb35 immunolabeling suggests that the ChAT fibers may be
interacting with mAb35-immunopositive fibers in the fiber tract lateral
to the SpL. Our mAb 35 immunohistochemistry results confirm findings of
mAb35 immunolabeling in the SpL previously reported in chicken (Swanson
et al., 1983; Ullian and Sargent, 1995). However, mAb35 also recognized
fibers in the cholinergic tract lateral to the SpL. The origin of the
mAb35-positive fibers is not clear, nor is it known whether they are
attributable to the presence of 5 versus 3 nicotinic receptor
subunits. They could be processes from SpL neurons and/or afferents to
the area that are also recognized by mAb35. We do not believe that the
mAb35 fibers are identical to the ChAT fibers because there does not
appear to be a one-to-one correspondence between them. In addition, in
sections containing the nucleus semilunaris that were double-labeled
with the mAb35 and ChAT antibodies, only ChAT labeling was present in
the nucleus semilunaris. Watson et al. (1988) have also reported
that 125I-mAb35 did not bind to the
nucleus semilunaris in the finch. Merged images of the double-labeled
sections suggested that the greatest overlap of ChAT fibers with the
mAb 35 immunoreactivity was in the lateral neuropil (Fig. 1). Images
obtained at higher magnification within the SpL demonstrated that the
ChAT-immunolabeled fibers were associated with mAb35-labeled fibers
rather than cell bodies. When serial optical sections at 1 µm
intervals in the z plane were obtained through individual SpL neurons,
it appeared that there was little interaction of ChAT fibers with the
surface of the somas; rather, the ChAT was associated with
mAb35-immunoreactive fibers. Some of the ChAT-immunopositive fibers
were associated with mAb35-immunopositive fibers that appeared to be
processes of SpL neurons (Fig. 2), but whether all of the mAb 35 positive fibers originated in the SpL could not be established.
If, as the immunohistochemical data suggests, the mAb35-positive
processes of SpL neurons are associated with ChAT afferents, we would
predict that direct nicotinic responses could be evoked after
stimulation of the cholinergic fiber tract. Indeed, after electrical
stimulation in the cholinergic tract, three classes of monosynaptic
responses could be distinguished with the appropriate antagonists:
GABAergic, glutamatergic, and nicotinic. These synaptic potentials
correspond to the known afferent projections to the SpL (Reiner et al.,
1982). When the inhibitory GABAA currents were
blocked with bicuculline, many of the SpL neurons exhibited an action
potential after afferent stimulation. The SpL neurons therefore appear
to be under a strong GABAergic inhibition. The GABAergic innervation
could be caused by afferents from the paleostriatum primitivum (avian
homolog of the globus pallidus) or the substantia nigra. It is also
possible that some GABAergic SpL neurons innervate each other (Veenman
and Reiner, 1994). The excitatory potentials recorded from the SpL
neurons were mediated by glutamatergic and nicotinic receptors as
evidenced by the blockade of the potentials by DNQX, AP-5, and DHE.
The cholinergic afferents, as has already been discussed, originate in
the nucleus semilunaris, and the glutamatergic afferents project from
the anterior ansa lenticularis, the avian homolog of the subthalamus
(Medina et al., 1997).
Stimulation of cholinergic afferents in the SpL produces a fast
excitatory nicotinic postsynaptic potential that can generate an action
potential. The isolated synaptic response is nicotinic because it is
blocked by nicotinic antagonists. In addition, blocking acetylcholinesterase with eserine altered the amplitudes and decay rates of the evoked response. Eserine also has direct effects on
nicotinic receptors at concentrations required to block the enzyme
(Mizobe and Livett, 1982; Shaw et al., 1985), and therefore the effects
of eserine on nicotinic neurotransmission can be complex. The
current-voltage relationship of the evoked nicotinic response behaves
as others have reported for nicotinic receptors in that the
extrapolated reversal potential is at 0.6 mV and the curve shows strong
rectification at positive holding potentials. All of these results lead
us to conclude that responses we recorded from SpL neurons were caused
by fast excitatory nicotinic neurotransmission.
To date only a few examples of evoked fast nicotinic neurotransmission
in the CNS are known. One is at hippocampal interneurons (Frazier et
al., 1998; Alkondon et al., 1998). The nicotinic neurotransmission at
hippocampal interneurons appears to be mediated by 7-containing nicotinic receptors because the response is blocked by 50-75
nM MLA. In contrast, the response we recorded from SpL
neurons is not blocked by 100 nM MLA and requires
concentrations of MLA (30 µM) that antagonize all classes
of nicotinic receptors to observe inhibition. We have previously shown
that somatic-dendritic nicotinic receptors on SpL neurons require
10-30 µM MLA for a blockade of activation by exogenous
nicotinic agonists, whereas other central and ganglionic nicotinic
receptors are blocked at significantly lower MLA concentrations (Yum et
al., 1996). Although Ullian and Sargent (1995) found that up to 20% of
SpL neurons expressed the 7 subunit, we did not observe evoked
responses that were sensitive to low concentrations of MLA. Therefore,
7-containing receptors do not appear to be mediating the observed
synaptic response and may be playing other functional roles in SpL
neurons. For example, 7 receptors may also be localized on SpL
terminals in the optic tectum in which they could modulate GABA
release. Alternatively, it may be that we have not sampled enough
neurons to detect the 7 receptor-mediated responses.
Another example of an evoked central nicotinic response is in the
visual cortex of the ferret (Roerig et al., 1997). The nicotinic responses in the ferret were blocked by DHE (50-100
µM) but not by -bungarotoxin (100 nM),
suggesting that they, like the responses presented here, are mediated
by a non-7-containing nicotinic receptor. Roerig et al. (1997) did
not examine the evoked response in the current-clamp mode, so it is
unclear whether the evoked nicotinic response could elicit action potentials.
The nicotinic receptors mediating the response in the SpL are
likely to be made up of combinations of the 2, 5, and/or 4 subunits with the 2 subunits. All of these subunits are found in
SpL, and the 2 and 5 subunits have the highest level of
expression at 18-19 d of incubation in the chick embryo (Daubas et
al., 1990; Ullian and Sargent, 1995). Most SpL neurons appeared
immunoreactive for mAb35 in the present study as well. The results of
three different experimental approaches have indicated that there are
no 3 subunits in SpL neurons, so the mAb35 binding is to the 5
subunit. Ullian and Sargent (1995) concluded that the mAb35 was binding
to the 5 nicotinic subunit because an antibody specific for the 3
subunit did not bind to SpL neurons. In situ hybridization
has not found mRNA for the 3 subunit in SpL neurons (Morris et al.,
1990), and there is very little
125I- -bungarotoxin binding,
indicating a lack of 3 nicotinic receptor subunits in the SpL
(Sorenson and Chiappinelli, 1992). Heterogeneity in these high-affinity
nicotine receptors exists, as indicated by multiple conductances
observed in cell-attached single channels recorded from SpL neurons
(Weaver and Chiappinelli, 1996) and by whole-cell recording that
revealed differences in sensitivities to the nicotinic antagonist
trimethaphan (Weaver et al., 1994). On the other hand, all of the
receptors exhibit very similar sensitivities to DHE (Weaver et al.,
1994). The cholinergic synapses mediating the evoked responses are
likely to be on the dendrites of the SpL neurons, because the
cholinergic fibers are associated predominantly with
mAb35-immunopositive fibers.
In conclusion, we report that non-7-containing nicotinic receptors
mediate fast EPSPs that can generate action potentials in SpL neurons.
The nicotinic cholinergic synapses appear to be on the dendrites of the
SpL neurons rather than on the neuronal somas. Therefore, the SpL can
be used as a model system in which to study the role of fast nicotinic
neurotransmission in regulating the activity of neurons in the CNS.
| |
FOOTNOTES |
Received April 19, 1999; revised June 18, 1999; accepted June 28, 1999.
This research was supported by National Institutes of Health Grant NS
17574 to V.A.C. We thank Miles L. Epstein for his generous contribution
of anti-ChAT antiserum.
Drs. Nong and Sorenson contributed equally to this work.
Correspondence should be addressed to Dr. Vincent A. Chiappinelli,
Department of Pharmacology, The George Washington University Medical
Center, 2300 Eye Street NW, Washington, DC 20037.
| |
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