1The Center for Neurobiology and Behavior and 2Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032
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ABSTRACT |
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Barazangi, Nobl and Lorna W. Role. Nicotine-Induced Enhancement of Glutamatergic and GABAergic Synaptic Transmission in the Mouse Amygdala. J. Neurophysiol. 86: 463-474, 2001. Presynaptic nicotinic acetylcholine receptors (nAChRs) are thought to mediate some of the cognitive and behavioral effects of nicotine. The olfactory projection to the amygdala, and intra-amygdaloid projections, are limbic relays involved in behavioral reinforcement, a property influenced by nicotine. Co-cultures consisting of murine olfactory bulb (OB) explants and dispersed amygdala neurons were developed to reconstruct this pathway in vitro. Whole cell patch-clamp recordings were obtained from amygdala neurons contacted by OB explant neurites, and spontaneous and evoked synaptic currents were characterized. The majority of the 108 innervated amygdala neurons exhibited glutamatergic spontaneous postsynaptic currents (PSCs), 20% exhibited GABAergic spontaneous PSCs, and 17% exhibited both. Direct extracellular stimulation of OB explants elicited glutamatergic synaptic currents in amygdala neurons. Antibodies to nAChR subunits co-localized with an antibody to synapsin I, a presynaptic marker, along OB explant processes, consistent with the targeting of nAChR protein to presynaptic sites of the mitral cell projections. Hence, we examined the role of presynaptic nAChRs in modulating synaptic transmission in the OB-amygdala co-cultures. Focal application of 500 nM to 1 µM nicotine for 5-60 s markedly increased the frequency of spontaneous PSCs, without a change in the amplitude, in 39% of neurons that exhibited glutamatergic spontaneous PSCs (average peak fold increase = 125.2 ± 33.3). Nicotine also enhanced evoked glutamatergic currents elicited by direct stimulation of OB explant fibers. Nicotine increased the frequency of spontaneous PSCs, without a change in the amplitude, in 35% of neurons that exhibited GABAergic spontaneous PSCs (average peak fold increase = 63.9 ± 34.3). Thus activation of presynaptic nAChRs can modulate glutamatergic as well as GABAergic synaptic transmission in the amygdala. These results suggest that behaviors mediated by olfactory projections may be modulated by presynaptic nAChRs in the amygdala, where integration of olfactory and pheromonal input is thought to occur.
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INTRODUCTION |
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The olfactory projection to
the amygdala, which includes projections from both the main and
accessory olfactory bulbs, is a limbic relay that has been implicated
in the reinforcement of stereotypical behaviors (Halpern
1987; Keverne 1999
; Mori et al. 1999
; Newman 1999
; Swanson and Petrovich
1998
) and in certain forms of learning and memory
(Granger and Lynch 1991
; Keverne 1995
;
Sullivan and Wilson 1993
). In addition, intra-amygdaloid projections have been implicated in reinforcement (Everitt et al. 1999
; Kalivas and Nakamura 1999
) and in
forms of learning and memory related to sexual behavior, fear, and
aggression (Aggleton 1992
; Fanselow and LeDoux
1999
; Gallagher and Chiba 1996
; Maren 1999
; Pfaus 1999
). To address the mechanism by
which reinforcement, learning, and memory may occur at the synaptic
level, we examined the synaptic transmission properties of the
olfactory bulb (OB)-amygdala and intra-amygdaloid projections.
Cholinergic neurons in the basal forebrain send projections to several
nuclei within the amygdala that also receive input from the OB
(Aggleton 1992; Arvidsson et al. 1997
;
Schafer et al. 1998
; Woolf et al. 1984
),
but their function remains unclear. Cholinergic projections to the OB
have been shown to be important in sensory processing at the synaptic
level (Alkondon et al. 1996
; Castillo et al.
1999
; El-Etri et al. 1999
) and have been
implicated in the susceptibility of olfactory function in Alzheimer's
disease (Durand et al. 1998
; Kasa et al.
1997
). Endogenous acetylcholine furnished by cholinergic
afferents to the amygdala may also modulate transmission at the
OB-amygdala and intra-amygdaloid synapses.
The contribution of nicotinic acetylcholine receptors (nAChRs) to
the cholinergic system in the amygdala is particularly interesting because nicotine, acting as a positively reinforcing stimulus, can
affect several cognitive functions, such as attention, learning, and
memory. Neuronal nAChRs are located in regions of the CNS that are
involved in these functions (Changeux et al. 1998;
Corrigall et al. 1992
; Levin and Simon
1998
; Picciotto et al. 1998
), and nicotine is
believed to exert these effects by interacting with other
neurotransmitter systems (Levin and Simon 1998
;
Miyata and Yanagita 1998
). The specific mechanisms by
which neuronal nAChRs may mediate reinforcement have undergone much
investigation (Dani and Heinemann 1996
).
Anatomical, neurochemical, and neurophysiological data suggest that
central nAChRs function as presynaptic modulators of synaptic transmission as well as direct mediators of synaptic transmission (MacDermott et al. 1999; McGehee and Role
1996
; Wonnacott 1997
). Activation of presynaptic
or preterminal nAChRs has been shown to enhance the release of many
neurotransmitters in various CNS regions (Alkondon et al.
1999
; Aramakis and Metherate 1998
; Gray et al. 1996
; Guo et al. 1998
; Lena and
Changeux 1997
; Lena et al. 1993
; Luo et
al. 1998
; McGehee et al. 1995
;
Pidoplichko et al. 1997
). The modulatory role of
presynaptic nAChRs in limbic regions that participate in reinforcement,
attention, and arousal is of particular interest, as the presence of
such receptors could be related to the addictive properties of nicotine
(Changeux et al. 1998
; Levin and Simon
1998
).
Several observations suggest that nAChRs may be localized to the
presynaptic terminals of the olfactory projection to the amygdala. mRNA
for nAChR subunits is expressed in the main and accessory olfactory
bulbs (Keiger and Walker 2000; Moser et al. 1996
; Seguela et al. 1993
; Wada et al.
1989
), and nAChR binding studies have revealed nAChR protein in
the amygdala (Hill et al. 1993
; Hunt and Schmidt
1978
). Hence endogenous acetylcholine furnished by cholinergic
afferents to the amygdala may modulate transmission at the OB-amygdala
synapse via presynaptic nAChRs.
We are interested in the potential contribution of the intra-amygdaloid and olfactory-amygdala limbic relays in positive reinforcement and integration of olfactory input. To this end, we examined the spontaneous and evoked synaptic transmission properties of these relays. As an intact brain slice preserving the projection of the OB to the amygdala cannot be prepared, we reconstructed the OB-amygdala synapse in vitro by preparing co-cultures of murine OB explants and dispersed amygdala neurons. The majority of TTX-resistant spontaneous synaptic transmission, and all stimulus evoked responses elicited by OB explant stimulation, were glutamatergic, suggesting that at least a portion of glutamatergic transmission is due to OB-amygdala synaptic interactions. Spontaneous GABAergic synaptic transmission was also detected in the presence of TTX, consistent with GABAergic synaptic interactions among amygdala neurons. The presence of putative presynaptic nAChRs and the endogenous ligand, acetylcholine, at olfactory projection terminals, led us to investigate the possible role of presynaptic nAChRs in modulating transmission in the co-cultures. We found that low concentrations of nicotine resulted in the facilitation of glutamatergic spontaneous and stimulus-evoked postsynaptic currents, as well as in the facilitation of GABAergic spontaneous postsynaptic currents.
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METHODS |
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Tissue culture
OBs were dissected from embryonic day 14 (E14) mouse pups removed from timed-pregnant animals killed
by exposure to carbon dioxide and cervical dislocation (C57/Black 6 timed-pregnant, Jackson Laboratory, Bar Harbor, ME). The OBs were cut
into pieces approximately 0.5-1 mm diam and thickness, and four to
five pieces were plated per plastic tissue culture dish, filled with 1 ml of media. The dishes were coated with poly-ornithine (2 µg/ml, Sigma, St. Louis, MO) and laminin (0.01 mg/ml, GIBCO, Rockville, MD),
and the media composition was as follows: DMEM including 5% fetal calf
serum, 5% horse serum (GIBCO, Rockville, MD), 8.3 mM dextrose (Sigma,
St. Louis, MO), 0.5 µM nerve growth factor (Harlan Bioproducts for
Science, Madison, WI), 100 units/ml penicillin, 100 µg/ml
streptomycin (GIBCO, Rockville, MD), 2 mM L-glutamine, 1 mg/10 ml human apo-transferrin, 0.5 mg/100 ml insulin, and 0.02 µM
progesterone (Sigma, St. Louis, MO). Two days after OB explants were
plated, amygdalas from E16 mice were removed using the
general techniques described for amygdala dissection (Arimatsu
et al. 1985; Cratty and Birkle 1994
;
Kasckow et al. 1997
; Lorenzo et al.
1992
). Briefly, a Leica Wild M3Z dissecting microscope (Leica, Deerfield, MI) was used at ×10 magnification to visualize the ventral
side of the brain and to identify the borders for dissecting the
amygdala. An approximately 2 mm by 2 mm region of the brain approximately 1-2 mm lateral to the hypothalamus, 1 mm from the lateral edge of the brain, 1-2 mm from the posterior edge of the brain, and extending from the ventral surface of the brain to the
lateral ventricle was dissected using micro dissecting spring scissors
and a scalpel under ×15 magnification. This amygdaloid region included
central, medial, lateral, and cortical nuclear groups, which include
nuclei that receive direct and indirect relays of the main and
accessory olfactory bulbs. The amygdalas were dissociated in 15 mg of
papain and 1.5 mg of cysteine (Sigma, St. Louis, MO) in 10 ml of
balanced salt solution (BSS) for 15 min. After three rounds of
trituration and washes with media, the amygdala neurons were plated
over the OB explants at a final density of 10,000-100,000 cells/dish.
The cultures were incubated at 37°C and 5%
CO2.
Immunostaining
Neurotensin staining was performed on sections of
E16 brains fixed with 4% paraformaldehyde for approximately
3 h at 4°C, after dissection from the skull. They were then
cryoprotected with a 30% sucrose solution and stored in Tissue
Freezing Medium (Triangle Biomedical Science, Durham, NC) at 80°C.
Sections (15 µm) were postfixed with 4% paraformaldehyde and washed
3 × 5 min with PBS containing 0.2% triton and 50 mM
NH4Cl (PBSTN). They were blocked with 10% goat
serum in PBSTN for 1 h at room temperature, and stained with
anti-neurotensin antibody (1:25-1:50 dilution, Research Diagnostics,
Flanders, NJ) 24 h at 4°C. The sections were then washed with
PBS containing NH4Cl (PBSN) 3 × 5 min, and a peroxidase-conjugated goat anti-rabbit secondary antibody (1:250, Jackson Immunoresearch, Westgrove, PA) in 10% goat serum, and PBSN was
used for 1 h at room temperature. The sections were co-stained with peroxidase using a VIP Substrate Kit for Peroxidase (Vector Laboratories, Burlingame, CA). OB explant cultures were stained with
antibodies at 4-6 days in vitro (DIV). They were fixed with 4%
paraformaldehyde and 4% sucrose for 20-30 min, permeabilized with
0.25% triton for 5 min, blocked with 10% bovine serum albumin (BSA)
for at least 30 min at 37°C, and treated with the primary antibody in
PBS and 3% BSA overnight at 4°C. The anti-
2 antibody (1:100-1:1,000 dilution) was developed by J. Lindstrom, and the anti-GAD-6 antibody (1:500 dilution) was developed by D. I. Gottlieb, and both were obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the National Institute
of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA. The anti-
4 (1:50-1:800 dilution, RBI, Natick, MA), anti-
7
(1:250-1:1,000 dilution, RBI, Natick, MA), anti-synapsin I
(1:500-1:1,000 dilution, Stressgen, Victoria, BC, Canada), and
anti-tau (1:1,000, Sigma, St. Louis, MO) antibodies were purchased
commercially. The anti-neurotensin antibody (see above) was used at
1:25-1:50 dilution. Secondary antibodies were applied for 1 h at
37°C after 3 × 10 min washes with PBS. For the anti-nAChR
subunit and anti-GAD-6 antibodies, the appropriate biotin-conjugated
secondary antibody was used (1:1,000, Jackson Immunoresearch,
Westgrove, PA) followed by neutr-avidin oregon green (1:1,000,
Molecular Probes, Eugene, OR). The secondary antibody used against the
neurotensin, synapsin I, and anti-tau primaries was rhodamine-redX goat
anti-rabbit (1:500, Jackson Immunoresearch, Westgrove, PA). After
washing the secondary antibodies 3 × 10 min, the explants were
coverslipped using Vectashield mounting medium with DAPI (Vector
Laboratories, Burlingame, CA). Controls for each staining were
conducted by incubating without the primary antibody and then staining
with the secondary antibody; unless otherwise stated, all controls did
not show any staining. The explants were viewed using a Zeiss Axioskop
fluorescent microscope, and pictures were taken with a CRI Real-14
digital camera using ImagePro (Media Cybernetics, Silver Spring, MD).
Recording
We recorded from co-cultures 4-6 days after plating
dispersed amygdala neurons (i.e., 6-8 days after OB explant plating). The intracellular solution used for whole cell recordings included (in
mM) 130 KCl (or 120 KGluconate and 10 KCl), 5 NaCl, 0.4 CaCl2, 1 MgCl2, 1.1 EGTA,
10 HEPES, 0.3 GTP, and 5 ATP (pH 7.2-7.4). For perforated patch
recordings, the intracellular recording solution contained (in mM) 125 CsCl, 5 CaCl2, 10 HEPES, 10 EGTA, and 2 ATP (pH
7.3). The extracellular recording solution contained (in mM) 135 NaCl,
5 KCl, 2.5 CaCl2, 1 MgCl2,
5 HEPES, and 10 glucose (pH 7.3). Patch pipettes were pulled at 2.5-5
M using KIMAX-51 glass capillary tubes (Kimble Products, Vineland,
NJ). Membrane currents were measured by the whole cell configuration of
the patch clamp (Hamill et al. 1981
) or with
amphoteracin-permeabilized patches (Horn and Marty
1988
). Unless otherwise stated, all recordings were conducted
in the voltage-clamp configuration with a holding potential of
Vh =
60 mV. Spontaneous postsynaptic
currents were recorded continuously using a List EPC-7 amplifier (List,
Darmstadt, Germany) and a video cassette recorder through a digital
interface (VR10B, Instrutech, Port Washington, NY), in the presence of
1-3 µM tetrodotoxin (TTX) at 33°C. They were digitized using
pClamp 7.0 software (Axon Instruments, Foster City, CA) at 5 kHz, and filtered at 1 kHz prior to analysis. A Master 8 stimulator connected to
a stimulation isolation unit (A.M.P.I., Jerusalem, Israel) was used for
the direct stimulation of OB explant projections. Evoked postsynaptic
currents were recorded using a HEKA amplifier and Pulse software (HEKA
Elektronik, Lambrecht, Germany). Extracellular electrical stimulation
of projections emanating from OB explants was conducted by applying
depolarizing pulses with concentric bipolar electrodes (FHC,
Bowdoinham, ME) or theta glass (WPI, Sarasota, FL) pulled at 3-5 µm
tip diameter and filled with extracellular solution. Evoked currents
were elicited by stimulating the OB explant projections for 200 µs at
0.1 Hz. Evoked postsynaptic currents were deemed monosynaptic if the
response was stimulus-locked and the delay after stimulation of the OB
explant fibers was <10 ms. Only monosynaptic evoked postsynaptic
currents, chosen by these criteria, were used for analysis.
Drug application
TTX (Calbiochem, La Jolla, CA),
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX),
D-5-aminopyridine (AP-V; Tocris, Ballwin, MO), nicotine,
bicuculline, acetylcholine, atropine (Sigma, St. Louis, MO), and
-bungarotoxin (Molecular Probes, Eugene, OR) were dissolved in the
extracellular solution at least 15 min prior to recording. The cells
were continuously perfused with the extracellular solution using bath
perfusion at approximately 1 ml/min. Nicotine and acetylcholine were
applied focally under visual control by pressure application (1-2 µm
tip diameter). Other drugs were applied via the bath perfusion.
Data analysis
Spontaneous postsynaptic currents were analyzed using the Mini Analysis Program (Synaptosoft, Leonia, NJ) and Microcal Origin (Microcal Software, Northampton, MA). The baseline frequency was determined by averaging the number of events in 10-s bins over at least 2 min prior to nicotine application. Decay constants were fitted using a scaled average and a double exponential using the Mini Analysis Program. The peak fold increase after nicotine application was determined by comparing the maximum number of events in one 10-s bin postnicotine to the average baseline frequency. The average fold increase was determined by comparing the average frequency during the first three 10-s bins after nicotine application to the average baseline frequency. A response to nicotine was determined to be significant by comparing the inter-event interval for 2 min before and after nicotine application, and then conducting an ANOVA test. Using these criteria, the lower limit for the fold increase in the spontaneous synaptic current frequency ± nicotine was approximately threefold. Hence this analysis may have underestimated the number of cells that exhibited lower magnitude responses. The evoked currents were analyzed using Pulse Fit (HEKA Elektronik, Lambrecht, Germany). Averages are stated as means ± SE.
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RESULTS |
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Establishment of synaptic interactions between olfactory bulb and amygdala neurons in vitro
To examine the electrophysiological properties of
the olfactory-amygdala synapses, we first determined
conditions under which both OB explants and amygdala neurons could be
maintained in vitro. Neurotensin was chosen as a mitral cell marker
because of its specific expression by OB mitral cells during early
development and synaptogenesis (E14 to the end of the 1st
postnatal week). Immunohistochemical studies in rat demonstrate a
neurotensin staining pattern of OB mitral cell bodies, lateral
olfactory tract (LOT) fibers and fibers innervating the amygdala with a
temporal pattern similar to neurotensin mRNA expression (Hara et
al. 1982; Kiyama et al. 1991
). Anti-neurotensin
antibody staining of a representative horizontal section of an
E16 murine brain demonstrates the general organization of
the olfactory projection to the amygdala (Fig. 1A). Mitral cell bodies and
fibers in the OB, fibers in the LOT, and fibers entering the amygdaloid
area are labeled by anti-neurotensin antibody (Fig. 1,
B-D).
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Conditions for establishing an OB-amygdala co-culture system were
determined in a series of experiments based, in part, on previously
described techniques for peripheral and CNS synaptic co-cultures
(McGehee et al. 1995; Role 1988
). OB
explants (approximately 1-2 mm diam) obtained from postnatal day
0 (P0), E18, E16, and E14 mice
were initially plated in a small amount of media to maximize explant
thinning, and the ability of the explants to adhere to the substrate
and extend neurites was determined. OB explants from E16-P0
animals either did not adhere to the substrate or failed to extend
processes. At E14, 85% of the explants plated on a
substrate composed of poly-ornithine and laminin grew processes after 1 day in vitro (DIV). At 5 DIV, the E14 explant neurites had
extended approximately 1.5-2.5 mm from the explant.
To determine whether the projections from the E14 mouse OB explants were furnished by mitral cells, the principle projection neurons in the OB, we immunostained explants plated alone at 4-6 DIV against neurotensin. The majority of fibers emanating from the explants (>90%) showed punctate staining along the whole length of the fibers (Fig. 2A). These observations suggest that the projection fibers in the OB-amygdala co-cultures arise from mitral cells.
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Neuronal survival from dispersed amygdala tissue removed at E18, P0, and P2 was very limited, and the cultures were overgrown by nonneuronal cells. The use of the mitotic inhibitor 5-fluorodeoxyuridine (40 µL/2 ml) 1-3 days after plating decreased the extent of the nonneuronal cell proliferation but did not substantially improve neuron survival. Dispersed amygdala neurons from E16 animals, however, survived successfully for at least 7 DIV at a plating density of 10,000-100,000 cells/dish. The E16 neurons grew extensive processes that were immunostained with anti-tau antibody (demonstrated in Fig. 2B) and were morphologically consistent with pyramidal neurons of the amygdala, with somata diameters ranging from 10 to 20 µm. Approximately 30% of the dispersed amygdala neurons in the co-cultures were immuno-positive for GAD-65 after 5 DIV (Fig. 2B), demonstrating that GABAergic interneurons intrinsic to the amygdala are supported by our tissue culture conditions.
Optimal plating efficiency and survival of amygdala neurons were obtained if OB explants were established 2-3 days prior to the addition of dispersed amygdala neurons. Patch-clamp recordings were obtained 4-6 DIV after amygdala neuron plating. At 4 DIV, most amygdala neurons appeared to be contacted by OB fibers within a 1- to 2-mm radius around the explant. Olfactory bulbs derived from heterozygous TgN(GFPU)5Nagy transgenic mice (Jackson Labs, Bar Harbor, ME) that express green fluorescent protein and wild-type amygdala neurons were also used to make co-cultures to facilitate the identification of OB fibers contacting amygdala neurons (Fig. 2C). These data led us to the identification of a specific set of conditions that optimize plating efficiency, survival, process outgrowth, and "ease" of electrophysiology assays of OB-amygdala co-cultures.
Synaptic transmission properties in the OB-amygdala co-cultures
To test whether functional synapses could be established in the
OB-amygdala co-cultures, we recorded from amygdala neurons at 4-6
days post-co-culture using the whole cell or perforated patch-clamp
configurations. We chose neurons that appeared to be contacted by
fibers emanating from the OB explant. At least 46% (total assayed was
234) of the neurons were innervated, as indicated by the presence of
spontaneous excitatory and/or inhibitory postsynaptic currents
(spontaneous PSCs) in 1-3 µM TTX. As the mitral cell projections are
glutamatergic (Ottersen and Storm-Mathisen 1984;
Quaglino et al. 1999
), we tested for glutamate
receptor-mediated transmission in the OB-amygdala co-cultures. In the
presence of TTX, a class of spontaneous PSCs that was blocked by 10 µM CNQX and 50 µM AP-V (100%; n = 12) and was
insensitive to 20 µM bicuculline was detected in 61%
(n = 66/108) of synaptically active neurons (Fig.
3, A and
B). This class of spontaneous PSCs exhibited fast kinetics,
with an average decay
of 3.01 ± 0.28 ms (n = 66; Fig. 3C). The average baseline frequency of these
spontaneous PSCs was 2.33 ± 0.99 (SE) events/10 s (Fig.
3D). In sum, this class of spontaneous, TTX-resistant PSCs
exhibited sensitivity to CNQX and AP-V, insensitivity to bicuculline,
and fast kinetics, consistent with glutamate receptor-mediated
transmission.
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To further test whether a presynaptic source of these glutamatergic synapses is the OB explants, evoked synaptic transmission studies were conducted. OB explant projections were stimulated and evoked, monosynaptic responses, defined as responses that were stimulus-locked with a delay <10 ms after stimulation, were recorded. In all amygdala neurons in which we recorded a fast, inward monosynaptic response, 10 µM CNQX and 50 µM AP-V diminished the evoked postsynaptic current (n = 7; Fig. 3, E and F). Furthermore, in all of the neurons tested, a slow GABAergic response was not observed, suggesting that the OB explants do not provide GABAergic input to the amygdala neurons. These findings provide direct evidence for glutamatergic input arising from OB explants and suggest that the spontaneous glutamatergic transmission detected in the co-cultures can arise from olfactory input as well as intra-amygdaloid synaptic interactions.
As GABAergic interneurons constitute a significant proportion of
neurons in the amygdala (Aggleton 1992) and our in vitro preparation (see Establishment of synaptic interactions between olfactory bulb and amygdala neurons in vitro), we tested
whether GABAergic synapses were also established in the co-cultures. In the presence of TTX, a class of spontaneous PSCs that was blocked by 20 µM bicuculline (83%; n = 5) and insensitive to 10 µM CNQX and 50 mM AP-V was detected in 20% (n = 22/108) of synaptically active neurons (Fig.
4, A and B). These
spontaneous PSCs exhibited slow kinetics, with an average decay
of
19.05 ± 1.98 ms, and the average baseline frequency was 0.82 ± 0.2 events/10 s (Fig. 4, C and D). In sum,
this class of spontaneous, TTX-resistant PSCs exhibited sensitivity to
bicuculline, insensitivity to CNQX and AP-V, and slow kinetics,
consistent with GABA receptor-mediated transmission. Studies of
stimulus-evoked transmission at OB-amygdala synapses revealed only
CNQX- and AP-V-sensitive evoked responses (see above). As such, the
GABAergic transmission in the co-cultures is likely a result of
amygdala-amygdala synaptic interactions. Finally, 17%
(n = 18/108) of cells exhibited both types of
spontaneous PSCs. The average decay
for fast spontaneous PSCs
exhibited by these cells was 4.35 ± 0.82 ms, the average decay
for slow spontaneous PSCs was 23.9 ± 5.15 ms, and the
baseline frequency for both types of spontaneous PSCs in these cells
was 3.35 ± 1.49 events/10 s. Two neurons that exhibited
spontaneous PSCs only after application of nicotine were also
considered to be innervated (see next section).
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Nicotine enhances glutamatergic and GABAergic transmission in the amygdala
Previous studies have shown that main OB and accessory olfactory
bulb mitral cells express mRNA for the 2,
3,
4,
5,
7,
2, and
4 nAChR subunits (Keiger and Walker 2000
;
Moser et al. 1996
; Seguela et al. 1993
;
Wada et al. 1989
). These findings, together with
numerous studies demonstrating the binding of nAChR agonists,
antagonists, and receptor subunit antibodies in the amygdala
(Arimatsu et al. 1978
; Hill et al. 1993
;
Hunt and Schmidt 1978
), are consistent with the
hypothesis that there may be presynaptic nAChRs expressed at terminals
of OB projections to the amygdala. To test whether E14 OB
explant fibers express nAChRs at their terminals, we immunostained
cultures with antibodies against the
4,
7, and
2 nAChR
subunits 4-6 days after plating. All three antibodies show staining
along the length of OB projections, although the distal axons and
axonal filopodia appear to be labeled more intensely (Fig.
5, A-C). We also examined
whether antibodies to the nAChR subunits and synapsin I, a presynaptic
marker, could co-label the OB explant projections. Co-localization of
each nAChR subunit antibody and the synapsin I antibody is demonstrated
at distal sites of the OB projection fibers (Fig. 5, D-F).
This suggests that nAChRs may be appropriately distributed to act as
presynaptic receptors at OB terminals.
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To test whether the activation of nAChRs might modulate synaptic transmission in the OB-amygdala co-cultures, we examined the effects of focally applied nicotine on the frequency of TTX-resistant, spontaneous PSCs. Synapses were determined to be glutamatergic based on their sensitivity to CNQX and AP-V, insensitivity to bicuculline, and fast kinetics (see previous section). Application of 500 nM to 1 µM nicotine significantly increased the frequency of glutamatergic, TTX-resistant spontaneous PSCs in 39% of the neurons studied (n = 33; Fig. 6, A and B). A representative response to nicotine is demonstrated in the histogram of synaptic current frequency shown in Fig. 6C. Nicotine's effect on the frequency of glutamatergic spontaneous PSCs was extremely robust. Figure 6D presents the pooled responses for all the glutamatergic synapses modulated by nicotine. The mean peak fold increase of glutamatergic spontaneous PSCs after nicotine application for all the neurons was 125.2 ± 33.3, while the mean fold increase (determined using the 1st three 10-s bins after nicotine application) was 55.6 ± 15. The average duration of the nicotine response for these cells, defined as the time until the spontaneous PSC frequency returned to baseline, was 125.8 ± 18 s, with a range from 16 to 380 s.
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To examine the potential role of nAChRs in the modulation of evoked glutamatergic transmission at the OB-amygdala synapse, we determined the effects of 1 µM nicotine on the failure rate and amplitude of evoked, CNQX- and AP-V-sensitive synaptic currents elicited by extracellular stimulation of the OB explant projections. In 24% of neurons tested with nicotine, a reversible decrease in the failure rate and/or a reversible increase of the amplitude of the stimulus-evoked postsynaptic current was observed (Fig. 6, E and F). These findings suggest that nicotine can directly modulate glutamatergic transmission at the OB-amygdala synapse.
The effects of nicotine on GABA-mediated transmission in the OB-amygdala co-cultures were also examined. Synapses were determined to be GABAergic based on their sensitivity to bicuculline, insensitivity to CNQX and AP-V, and slow kinetics (see previous section). Focal application of 500 nM to 1 µM nicotine elicited a significant increase in the frequency of GABAergic, TTX-resistant spontaneous PSCs in 35% of the neurons studied (n = 14; Fig. 7, A and B). A representative response to nicotine is demonstrated in the histogram of synaptic current frequency shown in Fig. 7C. Figure 7D presents the pooled responses for all the GABAergic synapses modulated by nicotine. The mean peak fold increase of GABAergic spontaneous PSCs after nicotine application for all the neurons was 63.9 ± 34.3, while the mean fold increase was 65.2 ± 29.3. In addition, the average duration of the response to nicotine was 150.8 ± 44.8 s, with a range of 20-630 s. It is notable that 2 of the 14 neurons that showed an increase in the frequency of GABAergic spontaneous PSCs did not have any detectable spontaneous PSCs prior to nicotine application.
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Increased inward current consistent with direct, postsynaptic effects of nicotine was detected in only 4% of amygdala neurons tested for nicotine-induced synaptic facilitation. The paucity of postsynaptic nAChR responses suggests that the nicotine concentration (500 nM to 1 µM) was likely too low to elicit a postsynaptic response, or that there may be a smaller number of postsynaptic nAChRs in the OB-amygdala co-cultures. Furthermore, changes in the amplitude of TTX-resistant, spontaneous PSCs were rarely seen following nicotine application (<5% of the neurons facilitated by nicotine). As such, the enhancement of spontaneous glutamatergic and GABAergic transmission in the OB-amygdala co-cultures is consistent with the activation of presynaptic nAChRs.
To assess the pharmacological properties of the nAChRs involved in
presynaptic facilitation, we compared the effects of acetylcholine (ACh) with those of nicotine on the modulation of glutamatergic synaptic transmission in the OB-amygdala co-cultures. ACh was applied
in a manner attempting to more closely correspond to delivery by
cholinergic projections in vivo [stimulation paradigm: 200-ms pulses
(5-7) of ACh (100-200 µM) applied at 0.2 Hz (Radcliffe and
Dani 1998)]. ACh elicited significant increases in the
frequency of TTX-resistant, spontaneous PSCs in approximately one-half
of the glutamatergic neurons tested (n = 7), as
elaborated in a representative frequency histogram (Fig.
8A).
Figure 8B presents the pooled responses for all the
glutamatergic synapses modulated by ACh. The mean peak fold increase of
the glutamatergic spontaneous PSCs after ACh application for all the
neurons was 13.42 ± 6.90, and the mean fold increase was
5.32 ± 2.70. The average duration of the ACh response for these
cells was 437.2 ± 80.1 s, with a range of 208-774 s.
Neither the holding current nor the amplitude of spontaneous,
TTX-resistant PSCs were significantly altered by ACh. These
observations suggest that ACh, like nicotine, primarily exerts a
presynaptic, rather than postsynaptic, effect on glutamatergic spontaneous transmission in OB-amygdala co-cultures. Experiments conducted in the presence or absence of 0.5 µM atropine, to eliminate the potential contribution of muscarinic acetylcholine receptors, were
equivalent, consistent with the activation of nAChRs under our
experimental conditions. In comparison to the magnitude of nicotine-induced enhancement of glutamatergic spontaneous PSC frequency, ACh elicited a significantly smaller increase in frequency (Fig. 8C).
|
Finally, pretreatment of the co-cultures with 100 nM alpha-bungarotoxin
blocked nicotine-induced facilitation of glutamatergic spontaneous
transmission. The mean fold increase of the glutamatergic spontaneous
PSCs after nicotine application for all the neurons tested was
17.82 ± 8.04 (n = 3), and the mean fold increase
after nicotine application in the presence of alpha-bungarotoxin was 0.83 ± 0.08 (n = 2). These results, in addition
to the observed differences between nicotine- and ACh-induced
facilitation, are consistent with the expression and localization of
7-containing nAChRs at sites of OB neuron projections. Thus
7-containing nAChRs may be involved in the nicotine-induced
facilitation of glutamatergic transmission at OB-amygdala synapses.
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DISCUSSION |
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The principle results of this study are the development of an in
vitro preparation suitable for study of synaptic interactions among
primary neurons of murine amygdala and OB. Explants of embryonic OB
were co-cultured with dispersed amygdala neurons in an attempt to
reconstruct the olfactory projection to the amygdala for direct visualization and analysis of synaptic interactions. Physical contact
between OB fibers and amygdala neurons developed within 4 days of
co-culture and functional synaptic interactions were detected by
patch-clamp recordings. At least 46% of the neurons were innervated by
4 days in vitro based on the detection of spontaneous, TTX-resistant
spontaneous PSCs. This number represents a lower estimate of the extent
of synaptic interactions as some neurons that appeared quiescent
received spontaneous synaptic input that was revealed only following
nicotine-induced facilitation. The majority (78%) of amygdala neurons
received glutamatergic input, and a smaller subset (37%) of neurons
received GABAergic input (including neurons that exhibited both types
of transmission). The level of spontaneous synaptic activity at
developing glutamatergic synapses, assessed as such by their
sensitivity to glutamate receptor antagonists (CNQX and AP-V), their
resistance to blockade by a GABA receptor antagonist (bicuculline), and
their fast kinetics, averaged 2.33 ± 0.99 events/10 s. GABAergic
spontaneous PSCs, which were isolated by their sensitivity to
bicuculline, insensitivity to CNQX and AP-V, and slow kinetics, were
significantly less frequent, averaging 0.82 ± 0.2 events/10 s.
Stimulation of the OB explant projections resulted in glutamatergic,
monosynaptic responses in the amygdala neurons, while there was no
evidence for stimulus-evoked GABAergic transmission at this synapse.
These data support our contention that glutamatergic synapses are
formed between OB explants and amygdala neurons in these co-cultures
and are consistent with the observation that mitral cells are
glutamatergic in vivo (Ottersen and Storm-Mathisen 1984;
Quaglino et al. 1999
). Thus it is likely that
glutamatergic TTX-resistant, spontaneous PSCs derive at least in part
from OB-amygdala synaptic interactions, in addition to synapses
between amygdala neurons.
A subset of neurons in the OB-amygdala co-cultures received GABAergic input (20% of innervated neurons). In contrast, one-half of the innervated neurons in dispersed cell cultures prepared from amygdala without extrinsic OB input received GABAergic synaptic input (data not shown). As evoked transmission studies did not reveal any GABAergic input arising from the OB explants, it is likely that the GABA-mediated synaptic transmission detected arises from interactions among neurons intrinsic to the amygdala. The relative rarity of GABAergic transmission in the OB-amygdala co-cultures is consistent with the lower plating density of amygdala neurons in these preparations. The lower incidence of GABAergic transmission is also consistent with a relatively low level of GAD-65-positive neurons in the co-cultures (approximately 30% of the neurons) versus the amygdala-only cultures (approximately 60% of the neurons).
Low concentrations of nicotine increased the frequency of glutamatergic
TTX-resistant, spontaneous PSCs in 39% of the neurons receiving
glutamatergic input, and of GABAergic spontaneous PSCs in 35% of the
neurons receiving GABAergic input. Nicotine similarly modulated evoked
glutamatergic transmission, elicited by the direct stimulation of OB
projections, resulting in an increase in the amplitude of evoked
responses and/or a decrease in the number of failures. Our
demonstration that activation of presynaptic nAChRs facilitates both
glutamatergic and GABAergic synapses in the amygdala provides further
support for a proposed modulatory role of cholinergic inputs and
nicotine-gated channels in the CNS (MacDermott et al.
1999).
Although prior studies have shown facilitation of both glutamatergic and GABAergic spontaneous PSCs in other CNS relays, the extent of nicotine-induced synaptic facilitation noted here is particularly striking. The >100-fold enhancement of glutamatergic transmission in OB-amygdala co-cultures likely translates into significant changes in synaptic strength. The robust response of OB-amygdala synapses to modulation by presynaptic nAChRs is most likely due to the number, localization, and composition of presynaptic nAChRs. The detection of coincident immunostaining of nAChR subunits and synaptic vesicle markers in the OB explant projections is consistent with the clustering and/or high levels of targeted expression of nAChRs in areas of transmitter release. The modulation of both glutamatergic and GABAergic inputs to neurons within the amygdala further supports the role of presynaptic nAChRs and cholinergic projections in the fine-tuning of both intrinsic and extrinsic inputs to amygdala neurons.
ACh also resulted in an increase in the frequency of glutamatergic
TTX-resistant, spontaneous PSCs in the co-cultures. Modulation by ACh,
like nicotine, is most likely due to the activation of presynaptic
nAChRs. Despite the high concentrations and "pulsatile" delivery of
ACh, the magnitude of the response to ACh is significantly smaller than
that to nicotine. As recombinant nAChRs containing the 7 subunit are
approximately five times more sensitive to nicotine than ACh
(Couturier et al. 1990
), the smaller magnitude of
ACh-induced enhancement of transmission may be due to the lower affinity of
7-containing nAChRs for ACh than nicotine. Furthermore, preliminary assays of sensitivity of the nicotine-induced facilitation of glutamatergic transmission to alpha-bungarotoxin also suggest that
7-containing presynaptic nAChRs may be involved. As the OB explants
express protein for the
7 nAChR subunit, and it has been shown that
functional
7-containing nAChRs are expressed in the OB
(Alkondon et al. 1996
; Castillo et al.
1999
), we propose that glutamatergic synapses formed between OB
and amygdala neurons in vitro and in vivo are modulated by
7-containing presynaptic nAChRs. These findings are also consistent
with previous studies that implicate
7-containing presynaptic nAChRs
in other limbic regions, such as the medial habenula-interpeduncular
nucleus relay and the hippocampus, in the modulation of excitatory
transmission (Gray et al. 1996
; McGehee et al.
1995
).
Olfactory projections to the amygdala that include efferents of the
main and accessory olfactory bulbs are implicated in the integration of
reinforcing stimuli as well as the consolidation and storage of
olfactory memories. Such projections may in turn influence
stereotypical behaviors related to reproduction, fear, and aggression
(Granger and Lynch 1991; Halpern 1987
;
Keverne 1995
, 1999
). Nicotine-induced
enhancement of glutamatergic and GABAergic synaptic transmission in the
amygdala provides a possible functional role for nAChRs and cholinergic
projections in the amygdala and olfactory system, as well as a
potential mechanism by which simple behaviors associated with olfaction
and olfactory memories can be modulated. In view of the unique
projection of accessory olfactory bulb efferents to the amygdala, the
role of cholinergic modulation in control of pheromone signaling should be examined in more detail.
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ACKNOWLEDGMENTS |
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We especially thank M. Jareb for extensive discussions and reading of the manuscript. We also thank R. Dammerman, W. Friedman, S. Gelber, R. Girod, A. MacDermott, and R. Yu for critical reading of the manuscript; A. Kriegstein for the use of laboratory equipment; and S. Siegelbaum and R. Axel for helpful discussions.
This work was supported by National Institutes of Health Grants NS-22061, DA-09366 (to L. W. Role), and NS-29832 (to S. Feinmark).
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FOOTNOTES |
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Address for reprint requests: L. W. Role, The Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, 1051 Riverside Dr., Psychiatric Institute Annex, Rm. 807, New York, NY 10032 (E-mail: lwr1{at}columbia.edu).
Received 20 October 2000; accepted in final form 9 March 2001.
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REFERENCES |
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