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REPORT
1Center for Neurobiology and Behavior, Columbia University College of Physicians & Surgeons; and 2Research Foundation for Mental Health, New York State Psychiatric Institute, New York, New York
Submitted 19 August 2007; accepted in final form 11 February 2008
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ABSTRACT |
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7, containing (7*) and non-7* nAChRs, facilitates glutamatergic transmission in an activity-dependent manner. Without prior stimulation, application of nicotine elicits modest and transient facilitation of glutamatergic postsynaptic currents (PSCs) in about 40% of BLA neurons. With low-frequency stimulation of cortical inputs nicotine elicits robust facilitation of transmission at about 60% of cortico–BLA synapses and synaptic strength remains elevated at about 40% of these connections for >15 min after nicotine washout. Following paired-pulse stimulation nicotine elicits long-lasting facilitation of glutamatergic transmission at about 70% of cortico–BLA connections. Nicotine reduces the threshold for activation of long-term potentiation of cortico–BLA synapses evoked by patterned stimulation. Prenatal exposure to nicotine reduced subsequent modulatory responses to acute nicotine application. |
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INTRODUCTION |
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). In the CNS, cholinergic projections are diffuse and nicotinic acetylcholine receptor (nAChR)–expressing target neurons are peppered through cortical, limbic, and central autonomic structures (Woolf 1991). Despite the widespread distribution of cholinoceptive sites, examples of direct nAChR-mediated fast synaptic transmission in the mammalian brain are relatively rare, possibly reflecting participation in functions other than the mediation of direct synaptic transmission per se (but see Alkondon and Albuquerque 2001, 2002; Klein and Yakel 2006). It has been proposed that ACh, released via "volume transmission," may act as a modulator of circuit excitability by interacting with pre-, post-, and perisynaptic nAChRs (Berg et al. 2006; Giocomo and Hasselmo 2005; Rousseau et al. 2005; Wonnacott et al. 2006; Zhang and Berg 2007). Indeed, numerous reports have documented the effects of presynaptic nAChR activation on the release of a variety of transmitters, including glutamate (Jo et al. 2005; Turner 2004; Wonnacott 1997; Zhang and Berg 2007).
The excitability of cortico–amygdala circuits, renowned for their role in consolidation of memories related to events of emotional salience, is modulated by cholinergic tone (Addy et al. 2003; Barros et al. 2005). In particular, the basolateral amygdala receives dense cholinergic inputs from the horizontal limb of the diagonal band nucleus and the ventral pallidum (Wainer et al. 1993; Woolf 1991). Vesicular acetylcholine transporter (VAChT) stained terminals are distributed throughout the amygdala with particularly high densities in the basal and lateral amygdala nuclei (Schafer et al. 1998).
Both 7* and β2* nAChRs are expressed in the basal and lateral amygdala nuclei (Hill Jr et al. 1993; Seguela et al. 1993). In rat basolateral amygdala, infusions of the 7* nAChR antagonists and the 4β2* nAChR antagonists impair working memory performance (Addy et al. 2003; Levin 2002). Likewise, increased cholinergic input to the basolateral nucleus of the amygdala (BLA) is associated with increased arousal, improved performance in short-term memory tasks, and enhanced acuity in discrimination of somatosensory inputs (Barros et al. 2005; May-Simera and Levin 2003).
The effects of nAChR activation on synaptic transmission per se have been examined in dispersed neurons from mouse amygdala (Barazangi and Role 2001) and in slice preparations from rat (Zhu et al. 2005). The current work tests the effects of nicotine at concentrations similar to those in moderate to heavy smokers on cortico–BLA transmission. We find that brief exposure to nicotine result in sustained, activity-dependent modulation of cortico–amygdala circuits and that prenatal exposure to nicotine results in long-lasting changes in the extent of cholinergic modulation. To the best of our knowledge, this is the first report of activity-dependent, nAChR modulation of cortico–amygdala circuits in wild-type (WT) versus genetically modified mice.
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METHODS |
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Coronal brain slices were prepared from C57BL6 backcrossed mice at postnatal age 14–28 days (Jackson Laboratory 7 genotype specifications). Mice used were +/+, +/–, or –/– for the 7 nAChR subunit gene. Animals were anesthetized with a mixture of ketamine and xylazine (100 mg ketamine and 6 mg xylazine/kg body weight injected intraperitoneally). After decapitation, the brain was transferred quickly into a sucrose-based ice-cold solution bubbled with 95% O2-5% CO2. This cutting solution contains (in mM): sucrose 230; KCl 2.5; MgSO4 10; CaCl2 0.5; NaH2PO4 1.25; NaHCO3 26; and glucose 10. Coronal brain slices (300 µm) were prepared using a Leica VT1000S Vibratome. Slices were equilibrated with an oxygenated artificial cerebrospinal fluid (aCSF) at room temperature (24–26°C) for 
1 h prior to transfer to the recording chamber. The slices were continuously superfused with aCSF at a rate of 2 ml/min containing (in mM); NaCl 126; KCl 2.5; NaH2PO4 1.25; NaHCO3 26; CaCl2 2; MgCl2 2; and glucose 10 bubbled with 95% O2-5% CO2 at room temperature (24–26°C).
In experiments on brain slices from older animals (e.g., >20 days), pyruvate (0.15–0.75 mM) was added to reduce oxidative damage and enhance survival (Gramsbergen et al. 2000; Matthews et al. 2003). With this protocol, slices are first incubated in a mixture of 50% cutting solution with pyruvate and 50% aCSF (in mM): sucrose 115; NaCl 63; KCl 2.5; NaH2PO4 1.25; MgSO4 5; CaCl2 1.25; MgCl2 1; NaHCO3 26; glucose 10; and sodium pyruvate 0.75 at 35°C for 30 min and then transferred to a mixture of 10% cutting solution and 90% aCSF (in mM): sucrose 23; NaCl 113.4; KCl 2.5; NaH2PO4 1.25; MgSO4 1; CaCl2 1.85; MgCl2 1.8; NaHCO3 26; glucose 10; and sodium pyruvate 0.15 at 35°C for 1–4 h prior to recording.
Electrophysiological recordings
BLA pyramidal neurons were visualized on an Olympus BX51WI upright microscope (Olympus Optical) equipped with differential inference contrast (DIC) optics. Patch electrodes with a resistance of 4–6 M
were pulled with a P-97 Brown Flaming electrode puller (Sutter Instrument). Signals were recorded with a Multi Clamp 700A amplifier (Axon Instruments). The pipette solution contains (in mM) 130 K-gluconate; 2 KCl; 2 MgCl2; 10 HEPES; 0.5 EGTA; 1 ATP; and 0.2 GTP (pH 7.3). The following criteria were applied for inclusion of recorded cells in the study; failure to meet all of these criteria resulted in exclusion from the sample population: 1) seal resistances maintained throughout the recording period at >5 G; 2) holding current in whole cell clamp configuration remained within 10% of the initial value and was 
100 pA; and 3) series resistance (Rs), measured every 10 min throughout the course of the experiment, remained stable (i.e., <10% change from initial value).
To examine transmission at cortical–BLA inputs, excitatory postsynaptic currents (EPSCs) were evoked by field stimulation with a concentric bipolar stimulation electrode (FHC) placed in the external capsule (Fig. 1 A, left). A 0.1-Hz single stimulation was delivered via the stimulation electrode. Stimulation strength was adjusted to trigger approximately 50% successful EPSCs. Paired-pulse modulation was examined over a range of stimulus intervals, varying from 50 to 200 ms. Pairs of stimuli were delivered at 0.1 Hz (interval 50 ms; from –50 to –150 µA). The paired-pulse modulation ratio (PPR) was defined as [(I2 – I1) x 100%]/I1. Where specified, a theta burst stimulation (TBS) pattern, in which four pulses of stimulation are given at 50 Hz and repeated 10 times at a 5-Hz interbust interval. Spontaneous EPSCs of BLA pyramidal neurons were monitored in voltage-clamp configuration, with a holding potential of –60 mV. Bicuculline (10 µM) was used to block 
-aminobutyric acid type A (GABAA) receptors, 50 µM 2-amino-5-phosphonovaleric acid (APV) was used to block N-methyl-D-aspartate (NMDA) receptors, and 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was used to block -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors. Miniature currents were recorded in the presence of 1 µM tetrodotoxin (TTX). Data were filtered at 2 kHz by Multi Clamp 700A and analyzed using pClamp9 (Axon Instruments) and Mini Analysis 6.0 (Synaptosoft). All experiments were done in the presence of 200 nM atropine, a concentration that blocks activation of muscarinic receptors without blocking nAChRs (Zwart and Vijverberg 1997).
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Prenatal nicotine exposure began at embryonic day 14 (E14) of gestation. Pregnant female mice were given drinking water containing either nicotine hydrogen tartrate salt (200 µg/ml) with sucrose (1%) or sucrose alone (control group). This dosing regimen continued from E14 through parturition and weaning. The effects of acute nicotine exposure were assayed in slice recordings with continuous superperfusion of aCSF plus local pressure application of nicotine at 100 nM to 1 µM. The optimal dose of 500 nM nicotine was chosen and was used in experiments shown, unless otherwise noted. Focal pressure application of drugs by a Picospritzer II (Parker Instrumentation) used 10 psi applied pressure for 2 to 3 min to a 2- to 3-µm-diameter patch pipette that was positioned about 15 µm from the soma of the recorded neurons.
Biocytin staining
In a subset of studies we recorded from BLA pyramidal neurons with patch pipettes containing 0.2–0.5% biocytin solution (Sigma). After completion of the recordings, slices were perfused with 4% paraformaldehyde + 0.2% picric acid + 0.1% glutaraldehyde (pH 7.4) for 10 min. Slices were then postfixed overnight at 4°C. After repeated washes with 0.1 M phosphate-buffered saline, slices were treated with 0.3% H2O2 for 10 min at room temperature and then incubated in 0.2% Triton X-100 and 0.2% albumin for 1 h at 4°C. Biocytin staining was visualized by peroxidase-coupled anti-avidin/biotin conjugation using an ABC kit (1:500, Pierce 32054) for 1 h at room temperature followed by incubation in 0.025% DAB plus 0.003% H2O2 Tris buffer solution for 15 min. Samples were mounted in Permount and relocalized and photographed with a CCD digital camera (DAGE-MTI) and DIC microscope.
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RESULTS |
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Morphological and electrical properties of BLA pyramidal neurons
The basolateral nucleus of the amygdala (BLA) is easily identified within coronal brain slices by the landmark "forking" appearance of the external capsule (EC) under DIC optics (Fig. 1A, middle). BLA neurons with pyramidal-shaped somas and evident apical dendrites were chosen for electrophysiological study (Fig. 1A, right). In initial experiments, biocytin (0.5%) was included in the patch pipette solution, so that recordings could be followed by immunostaining to determine basic features of neuronal morphology and the precise location of the recorded neuron within the BLA. Depolarizing current steps of 1.5-s duration were delivered to the pyramidal neurons to elicit action potentials. BLA pyramidal neurons included two major groups based on the sub- and superthreshold response profiles. The majority of pyramidal neurons (
90%) fire with moderate adaptation in action potential rate during a maintained depolarizing step (Fig. 1B, left). About 10% of neurons subsequently identified as pyramidal neurons had a strongly adapting action potential firing profile (Fig. 1C, left). Morphological analysis of biocytin-labeled neurons established that neurons with a moderate spike adaptation had extensive perisomatic dendritic arbors (Fig. 1B, right), whereas strongly adapting neurons had less branched perisomatic dendrites (Fig. 1C, right).
Baseline synaptic transmission in the BLA
Recording in identified BLA neurons with K+-gluconate patch solution and holding at –60 mV revealed ongoing spontaneous synaptic events of both polarities and varying amplitudes and kinetic profiles (Fig. 2 A, left). Application of the GABAA-receptor antagonist bicuculline eliminated the slower, outward synaptic currents; the remaining inward synaptic currents were rapid in both rise and decay time course (Fig. 2, A, middle; B and C, bicuculline). Recordings in the continued presence of bicuculline revealed a net disinhibition of faster spontaneous synaptic events (Fig. 2, B and C, bicuculline) and the addition of NMDA and AMPA receptor antagonists (see METHODS) abolished the bicuculline-resistant currents (Fig. 2, A, right, B, and C). These findings are consistent with the idea that the majority of fast synaptic transmission detected under these recording conditions in the BLA involves activation of ionotropic GABA and glutamate receptors.
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Activation of nicotinic receptors facilitates glutamatergic transmission in the BLA. The effects of a range of low concentrations of nicotine (from 100 nM to 1 µM) were initially assessed using either focal application or bath application as described under METHODS. Both spontaneous synaptic activity (data not shown) and TTX (1 µM)-resistant synaptic transmission were enhanced by local application of nicotine in 40% (16/40) of BLA neurons tested (Fig. 3, A and B). Enhancement of glutamatergic transmission was reflected in an increase in miniature postsynaptic current (mPSC) frequency (Fig. 3, C and F) without alteration of mPSC amplitude (Fig. 3D). Thus interevent intervals were significantly reduced [Fig. 3C, Kolmogorov–Smirnov (KS) test, P < 0.001, n = 16] without a change in amplitude (Fig. 3D, KS test, P = 0.7, n = 16). Amplitude histograms of the mPSCs were well fit by lognormal distributions with a unit mode of 8.8 ± 0.2 pA (r2 = 0.96; 
2/df < 0.0001; Fig. 3H). The observed increases in mPSC frequency without altered PSC amplitude are consistent with a presynaptic mechanism contributing to nicotinic facilitation.
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7* and non-7* type nAChRs contribute to short-term synaptic facilitation
Examination of the relative contribution of 7* versus non-7* nAChR was tested using both pharmacological and genetic deletion techniques. First, we monitored nicotine- induced facilitation of TTX-resistant glutamatergic synaptic transmission with and without nicotine or the selective 7* receptor antagonist -bungarotoxin (-BgTx). In the presence of 100 nM -BgTx, increased mPSC frequency by nicotine was observed in only 1/4 recordings (Fig. 3, E, F, and G, Table 1). The 4β2*-selective nAChR antagonist dihydro-beta-erythroidine (DHβE, 5 µM) was similarly effective; detectable facilitation occurred in <1/4 recordings (Fig. 3, E, F, and G, Table 1). The nonselective nAChR antagonist mecamylamine (100 µM) abolished all facilitatory effects of nicotine (Fig. 3, E, F, and G, Table 1).
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). Although the current study examined 100- to 1,000-fold lower concentrations of agonist with the goal of testing "smokers' concentrations" of nicotine, we did observe small (5- to 10-pA) shifts in the steady-state current during the application of nicotine in about 40% of recordings. A comparison of the -BgTx, DHβE, and mecamylamine sensitivity of the direct inward currents with that of nicotine-elicited facilitation revealed a similarly mixed profile of both 7* and non-7* nAChRs. Likewise, assay of nAChR-stimulated facilitation in slices from 7 mutant mice—by either nicotine application or by treatment with the acetylcholine esterase inhibitor ambenonium (500 nM)—confirmed involvement of both 7* and non-7* nAChRs in the modulatory effects (Tables 1 and 2).
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The TTX-resistant glutamatergic mPSCs recorded reflect inputs to the BLA neurons from several different sources that may (or may not) have different profiles of nAChR-mediated modulation. To examine the effects of nAChR activation on specific inputs to BLA pyramidal neurons, we stimulated cortical fibers within the external capsule, at the point that the tract bifurcates along the lateral and basolateral nucleus. Stimulus-evoked synaptic current responses were recorded in whole cell voltage clamp of 129 BLA pyramidal neurons. In the presence of bicuculline, stimulation of cortical inputs evoked PSCs with rapid rise (
1 ms) and decay ( 3–5 ms) kinetics. Modulation of stimulus-evoked PSCs by nicotine was examined by decreasing the magnitude of the stimulus delivered to the external capsule so that about 50% of stimuli elicited postsynaptic currents (Fig. 4 A, control). Under these conditions, a single application of nicotine (500 nM; 2 min) increased both the success probability and the average amplitude of the stimulus-evoked PSCs (Fig. 4, B1 and B2). Nicotinic facilitation of evoked transmission was significantly increased in 68% (35/51) of the BLA neurons tested with single stimuli. In contrast to the transient effects of nicotine on spontaneous transmission, the facilitation of evoked cortico–amygdala transmission was often sustained well after the washout of nicotine from the perfusion, persisting for >10 to 30 min (see Table 2).
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Modulation of paired-pulse evoked glutamatergic transmission at cortico–BLA synapses
To further examine the pre- vs. postsynaptic loci of nicotine-induced facilitation, we analyzed glutamatergic transmission at cortico–BLA synapses evoked by paired-pulse stimulation (PPS). Responses to paired stimuli were tested over a range of interstimulus intervals (ISIs) from 50 to 200 ms (Fig. 5 A), and stimulation strength was adjusted such that the first pulse of each pair evoked stimulus-locked responses. Although all ISIs tested elicited paired-pulse facilitation, the 50-ms interval appeared the most robust (Fig. 5, B, C, and D). As such, subsequent experiments used a 50-ms interval. The effects of nicotine on paired-pulse evoked responses were assessed at 23 cortico–amygdala inputs in 10 different slice preparations. Nicotine increased the total integrated current at 57% and elicited a sustained facilitation at 69% of the synapses. PPS of cortical inputs coupled with nicotine treatment facilitated synaptic responses for >30 min, longer than ever observed with single stimuli (Table 2). Profiles of the two major types of paired-pulse evoked responses observed before and after nicotine application are presented in Fig. 6, A and B and Fig. 6, C and D, respectively. The majority of PPS responses were facilitated in a sustained manner and synaptic enhancement was manifest as a greater increase in the amplitude of the second (as opposed to the first) postsynaptic current response (Fig. 6, A and B). Short-term facilitation was seen in about 30% of the nicotine-responsive synapses (e.g., in Fig. 6, C and D), with a larger effect of nicotine on the first PSC of the PPS responses. Analysis of all PPS recordings with nicotinic facilitation revealed that nicotine modulated the paired-pulse ratio (PPR) by >10% in the majority of pairs tested (11/13, Fig. 6, E and F) with a net average increase in synaptic strength (based on amplitude and total integrated current, Fig. 6, G and H) of about 35%. These data are also consistent with the involvement of a presynaptic mechanism in nicotine-induced synaptic facilitation.
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Stimulation of cortical inputs to the BLA with a theta burst stimulation (TBS) pattern (see METHODS) elicited brief (<10 min) versus sustained potentiation (>10 to 90 min) in about half of the pairs tested (Fig. 7, A and B). From a total of 16 cortico–BLA pairs, TBS elicited brief potentiation in 7 pairs and sustained potentiation in 8 pairs (TBS failed to potentiate one of the tested pairs). When 4 of the 7 pairs that were only transiently potentiated with TBS alone were exposed to a single application of nicotine, subsequent TBS elicited in sustained potentiation. That is, nicotine paired with TBS facilitated glutamatergic transmission for as long as the recordings could be maintained (90 min, Fig. 7C). For the one pair that was unaffected by TBS alone, the combination of TBS + nicotine administration elicited a transient potentiation. In the 8 pairs in which long-lasting potentiation was elicited by TBS itself, addition of nicotine did not elicit any further potentiation.
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The preceding experiments are consistent with the idea that the efficacy of nicotine facilitation is related to the level of synaptic activity at glutamatergic cortico–BLA inputs. In view of these findings and the results of prior studies on prenatal nicotine effects on amygdala-related behaviors, we examined whether exposure to nicotine in the pre- and perinatal periods led to long-term changes in cortico–amygdala circuits.
Six days of prenatal nicotine exposure and the same dosing regimen continued through weaning, followed by recording at postnatal days 15–22 revealed a broadening of the (nonnormal) distribution of mPSC frequency compared with control littermates. However, statistically significant changes in baseline glutamatergic transmission were not observed.
Subsequent assays of the nicotinic modulation of cortico–BLA transmission in prenatal nicotine versus control mice revealed significant effects. Miniature PSCs recorded under control conditions and following acute nicotine application in prenatal nicotine-treated animals are shown in Fig. 8, A and B. The frequencies of mPSCs recorded under control conditions were similar regardless of prenatal nicotine exposure. However, subsequent challenge with an acute application of nicotine facilitated spontaneous transmission in half as many BLA neurons from prenatal nicotine-treated versus control animals. Furthermore, the nicotine-elicited facilitation that was detected in prenatal nicotine-exposed animals was less than that in untreated siblings (Fig. 8, C and D, solid line, control animal control; broken line, control animal with acute nicotine; solid circle, prenatal exposed animals control; open circle, prenatal exposed animals with acute nicotine). Figure 8C shows the cumulative probabilities of interevent intervals illustrating that spontaneous transmission in control versus prenatal nicotine-exposed animals was similar but not identical. In amygdala slices from animals exposed to prenatal nicotine, facilitation of transmission by an acute application of nicotine was considerably less than that seen in controls but still significant (KS test, P = 0.004, n = 6). No statistically significant changes in direct, nicotine-elicited currents were seen in prenatal nicotine-treated versus untreated controls (Fig. 8, E and F).
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DISCUSSION |
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7* mutant mice were assessed to delineate the effect of low-level nicotine exposure on glutamatergic inputs to BLA neurons in mouse. Basic characteristics of BLA neurons in mouse appear similar to those described in the rat (Sah et al. 2003). Most BLA pyramidal neurons fire action potentials with relatively little spike adaptation; <10% displayed strongly adapting spike profiles in response to prolonged depolarizing current injection. The latter neurons, which also appeared to have a less elaborate profile of primary dendritic arborization, were not included in subsequent analysis. Our studies focused on cortico–amygdala glutamatergic synaptic transmission by examining the bicuculline-resistant postsynaptic currents. The bicuculline-resistant synaptic activity was deemed to be largely, if not exclusively, glutamatergic because all currents were blocked by a cocktail of AMPA and NMDA receptor antagonists.
Prior tract tracing and immunohistochemical studies have demonstrated that the major cholinergic input to the rodent BLA is from basal forebrain (Carlsen et al. 1985; Nagai et al. 1982; Woolf and Butcher 1982). Despite robust ACh-positive projections, primary cholinergic neurons are not present in the BLA (Schafer et al. 1998). Prior studies in rat have demonstrated a direct postsynaptic response of BLA neurons to relatively high concentrations of nAChR agonists. Most nicotinic effects are consistent with nAChR-mediated modulation of transmission, although direct cholinergic synaptic connections are also indicated by recent reports (Alkondon et al. 1998; Klein and Yakel 2006; Zhu et al. 2005).
The current studies focused on the effects of low ("smokers' level") concentrations of nicotine on inputs to BLA neurons to examine the possible modulatory role of acute and prenatal nicotine receptor activation in the developing BLA. In the presence of bicuculline, TTX and atropine activation of nicotinic receptors increased the frequency, but not the amplitude, of spontaneous glutamatergic PSCs, consistent with the involvement of presynaptic nAChRs.
Pharmacological studies comparing the effects of nicotine in the presence of selective nAChR antagonists and assessing nicotine-induced facilitation of cortico–amygdala transmission in WT versus 7* nAChR mutant mice indicate that both 7* and (4β2)* nAChRs contribute to the modulatory effects of low-level nicotine in the BLA. These findings, considered in the context of elegant behavioral studies of Levin and colleagues (Addy et al. 2003; Levin 2002; Levin et al. 2006), support the contention that nAChR activation may constitute an important mechanism for the modulation of contextual learning associated with fearful stimuli.
Changes in the strength of synaptic connections are fundamental to the process of information storage with activity-dependent modulation of pre- and postsynaptic components as key mediators of such plasticity. The input from somatosensory cortical areas provides the amygdala with highly detailed sensory information pertinent to discrimination via cortico–amygdaloid projections to the lateral and basolateral nuclei. Rapid, less finely tuned input from thalamic areas provides information related to gross stimulus discrimination (Armony et al. 1997).
Modulation of cortical inputs to the BLA by stimulation of the external capsule to evoke PSCs could arise from either single- or multiple-fiber inputs to the BLA. If originating from multiple fibers, a facilitation of release would be predicted to manifest in both increased release probability and increased amplitude of evoked responses. To decrease the contribution of activating multiple cortical afferents, we used a minimum stimulation paradigm (Stevens and Wang 1995). Single-fiber activation is indicated by the predominance of single quantal-coefficient evoked amplitudes in the response histogram profiles. With minimum stimulation, single evoked EPSCs showed an increase in success probability but unchanged mean amplitudes (not including failures). In view of the consistent value of the quantal size of about 9 pA before, during, and after nicotine application, it is unlikely that alterations in postsynaptic responsiveness to glutamate contribute to the observed nicotinic modulation (Korn and Faber 1998).
Paired-pulse stimulation (PPS) has been widely used as an assay of synaptic plasticity and the relative contributions of pre- and postsynaptic mechanisms (Jiang et al. 2000). Alterations in the PPR are consistent with changes in synaptic strength due, at least in part, to changes in presynaptic function. PPS studies of cortico–amygdala synapses revealed an overall increase in synaptic efficacy following nicotine treatment. An increased PPR following nicotine, the predominant observed effect, reflected a greater increase of peak 2 (P2) relative to peak 1 (P1). Decreased PPR was typically due to a greater increase in amplitude of the first peak relative to the second. In a minority of cases (4/13) the amplitude of both P1 and P2 increased proportionally, so that there was no change in PPR. The latter result underscores that a contribution of a postsynaptic component to the overall effect of nicotine on facilitating glutamatergic transmission cannot be ruled out.
We found that the extent and duration of nicotine-induced facilitation of cortico–BLA synapses was directly related to the pattern and intensity of stimulation of the cortical afferents. Without prior stimulation, nicotine elicited a moderate facilitation of mini PSCs. Forty percent (16/40) of neurons showed facilitation, but the facilitation was never sustained after nicotine removal and washout. With minimal stimulation of cortical inputs at a low frequency (0.1 Hz), nicotine elicited considerably more robust facilitation at about 60% of cortico–amygdala synapses, with 50% showing sustained facilitation that outlasted the nicotine administration by 10 min. With stronger PPS of cortico–BLA synapses, nicotine facilitates about 60% of the inputs and elicits a sustained facilitation at 70% of cortico–BLA synapses tested. The sustained facilitation elicited by PPS often lasted longer than that detected with nicotine plus single shock stimulation of cortical inputs (>30 min).
The activity dependence of the extent of nicotine-induced facilitation is even observed with a subset of synapses that are below threshold for long-term potentiation in response to theta burst stimulation (TBS) alone. The addition of nicotine to TBS was sufficient to convert transient, post-TBS potentiation to long-lasting facilitation of glutamatergic transmission. Nicotinic modulation of synaptic transmission is thought to be mediated, at least in part, by Ca2+ influx through activated presynaptic nAChRs. Activity-dependent modulation also causes presynaptic Ca2+ accumulation. The Ca2+ accumulated by repetitive activity in the presynaptic terminal could therefore be amplifying the nicotinic modulation.
Current evidence in the hippocampus indicates a role for postsynaptic changes during potentiation including increased number of synaptic AMPA receptors and/or an increase in single-channel conductance. However, there is also evidence that synaptic facilitation and depression in hippocampus can involve alterations in presynaptic function. Such changes are thought to arise from enhanced probability of release or in the quantity of glutamate released as a result of an alteration in fusion pore kinetics or quantal content.
Nicotine administration has long been associated with enhancement of attention, short-term memory function, and, in some cases, more prolonged aspects of cognitive processing. We tested the hypothesis that activation of nicotinic receptors at cortico–amygdala synapses might contribute to such effects of nicotine. The observation that the degree of modulation and the duration of nicotine-induced synaptic facilitation are related to the activity of cortico–amygdala inputs is, to the best of our knowledge, without precedent.
Nicotine readily crosses the placental barrier (Luck et al. 1985) and fetal exposure to nicotine damages the developing brain (Mansvelder et al. 2006). Prenatal exposure to nicotine increases the risk for cognitive deficits, attention deficits, behavioral problems, and learning disabilities. Several studies have shown that nicotinic receptor expression levels and inactivation profiles could be changed by prenatal nicotine exposures. Prior studies of the effects of prenatal nicotine exposure on postnatal CNS transmission revealed dramatic increases in baseline GABAergic transmission in appetite-related circuits (Jo et al. 2005). The effects of prenatal nicotine exposure on postnatal glutamatergic inputs to BLA appear relatively modest, but the subsequent modulation of cortico–amygdala synaptic transmission by nAChR activation is virtually abolished in prenatal nicotine-exposed animals.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of L. Jiang and L. Role: Department of Neurobiology and Behavior, SUNY, Stony Brook, NY 11794.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. Jiang, Center for Neurobiology and Behavior, Columbia University College of Physicians & Surgeons, 630 West 168th Street, New York, NY 10032 (E-mail: Lj2115{at}columbia.edu)
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ABSTRACT
INTRODUCTION
, control; 
, + nicotine). Control vs. + nicotine mPSCs are significantly different at P < 0.001 [Kolmogorov–Smirnov (KS) test]. D: cumulative distribution of the mPSC amplitudes from the 16 separate experiments in which nicotine elicited significant effects on mPSC frequency. The cumulative distribution of mPSC amplitudes was right-shifted with nicotine. Control vs. + nicotine mPSCs are not significantly different in this or other +/– nicotine comparison groups (
) and second (P2, 
) pulse of each pair from the recorded neuron shown in C.
