Introduction

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Hippocampal nicotinic acetylcholine receptors (nAChRs) are important target sites of the well known behavioral and cognitive effects of the tobacco alkaloid nicotine (Dani and Heinemann, 1996; Lloyd and Williams, 2000). In addition they also serve as the potential molecular target of endogenous acetylcholine (ACh) released from varicose axon terminals of septal cholinergic afferents (Frotscher and Leranth, 1985) acting on different subtypes of muscarinic and nAChRs (Lukas et al., 1999) via synaptic and nonsynaptic interactions (Vizi and Kiss, 1998; Vizi and Lendvai, 1999; Vizi, 2000).

The presence of nAChRs in the mammalian hippocampus has been revealed by molecular biological and immunocytochemical studies (Deneris et al., 1988; Freedman et al., 1993; Seguela et al., 1993). Hippocampal nAChRs play an important role in the regulation of noradrenergic neurotransmission (Sacaan et al., 1995, 1996; Clarke and Reuben, 1996; Sershen et al., 1997; Vizi and Lendvai, 1999), can stimulate [³H]serotonin release from the dissected slices (Lendvai et al., 1996), and may participate in the modulation of glutamate release (Gray et al., 1996). In addition, a series of electrophysiological studies indicate that the release of -aminobutyric acid (GABA) is also regulated by nAChRs in the hippocampus. Application of nAChR agonists directly excites hippocampal interneurons, appearing as a depolarization detected by whole-cell patch-clamp or as a hyperpolarization in recordings of hippocampal neurons (Reece and Schwartzkroin, 1991; Alkondon et al., 1997, 1999; Albuquerque et al., 1998; Frazier et al., 1998a; McQuiston and Madison, 1999). Recently, it was found that hippocampal GABAergic interneurons are responsive to the activation of at least two subtypes of nAChR, assembled from 4 and 2; and homopentameric 7 subunits, respectively (Alkondon et al., 1997, Jones and Yakel, 1997; McQuiston and Madison, 1999). 42 subtype is predominantly localized on the terminal or preterminal region, whereas 7-type homopentameric nAChRs are located on the somatodendritic region of interneurons, and mediate fast synaptic transmission (Alkondon et al., 1998; Frazier et al., 1998b); moreover, nicotine-responsive interneurons exhibit uneven cell-type-specific regional distribution (McQuiston and Madison, 1999). The many types of hippocampal interneurons show a great variety of morphological and functional diversity (Freund and Buzsáki, 1996; Vizi and Kiss, 1998), and they play different roles in the ensemble of hippocampal neuronal network, e.g., they can control behavior-dependent electrical activity patterns (Ylinen et al., 1995), synaptic plasticity (Maccaferri and McBain, 1995), and synchronization of large populations of principal cells at slow and fast frequencies (Cobb et al., 1995; Whittington et al., 1995) underlying different aspects of memory formation. Therefore, to understand the precise site of effect of memory enhancement by nicotine it is of crucial interest how nicotinic receptor activation influences the release of GABA from the interneurons of the hippocampus.

Early neurochemical studies indicated that nicotine enhanced 4-amino-n-[2,3-³H]butyric acid ([³H]GABA) release from rat hippocampal synaptosomes (Wonnacott et al., 1989) and the regulation of GABA release by nicotinic receptors has been described in other regions such as globus pallidus and substantia nigra (Kayadjanian et al., 1994) and guinea pig cortical slices (Bianchi et al., 1995), although these latter effects proved to be indirect. More recently, Lu et al. (1998) pharmacologically characterized hippocampal nicotinic receptors present on the nerve terminals derived from different brain regions, including the hippocampus. This study identified the 42 assembly as the major subunit composition responsible for stimulation of GABA release from mouse brain synaptosomes. However, there are no neurochemical data on the impact of nicotine receptor stimulation on GABA release in a more integrated system, i.e., in brain slices, and this enables the study of the effect of not only presynaptic but also preterminal and somatodendritic receptors on the release. These receptors may be activated by ACh released from both synaptic and nonsynaptic varicosities and by nicotine inhaled during smoking.

Therefore, in this report, we have directly measured the spontaneous and the electrically evoked outflow of [³H]GABA from rat hippocampal slices in in vitro release experiments, and explored the effect of nicotinic receptor stimulation. We show that the activation of 7-type nicotinic receptors of hippocampal interneurons results in a long-lasting ability of these cells to respond to depolarization with an increased release of GABA mediated by the transporter system. In addition the excitatory effect of nicotine on intracellular Ca²⁺ level measured from identified CA1 interneurons is also demonstrated.

	Materials and Methods

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[³H]GABA Release from Hippocampal Slices. [³H]GABA release experiments were carried out with slight modifications of the technique described in our previous studies (Katona et al., 1999; Vizi and Sperlágh, 1999). Male Wistar rats (140-160 g, breeder; Gedeon Richter Ltd., Budapest, Hungary) were decapitated and the brain was quickly put into ice-cold Krebs' solution of the following composition: 115 mM NaCl, 3 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 25 mM NaHCO₃, and 10 mM glucose. The solution was oxygenated with 95% O₂ and 5% CO₂, and the pH was 7.4. Both hippocampi were rapidly dissected and 400-µm-thick slices were cut transversely with a McIlwain tissue chopper and incubated in 1 ml of oxygenated Krebs' solution containing 4 µCi of [³H]GABA, specific activity 86.0 Ci/mmol (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, England) for 60 min at 37°C. The incubating solution was supplemented with -alanine (1 mM; Tocris Cookson Ltd., Bristol, England) to prevent tritium uptake into glial cells (Iversen and Kelly, 1975). After incubation, the slices were rinsed three times with 6 ml of Krebs' solution, and four slices were transferred to each of four polypropylene tissue chambers, and perfused continuously with 95% O₂- and 5% CO₂-saturated Krebs' solution (37°C, flow rate of 0.6 ml/min). To minimize the formation of GABA metabolites, the perfusion solution contained aminooxyacetic acid (100 µM; Sigma Chemical Co., St. Louis, MO). Upon termination of the 50-min preperfusion period, (±)-2-amino-5-phosphonopentanoic acid (AP-5) (10 µM; Research Biochemicals International, Natick, MA), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) disodium (10 µM; Research Biochemicals International), and atropine sulfate (10 µM; EGIS, Budapest, Hungary) were added to the perfusion solution to block excitatory N-methyl-D-aspartate (NMDA)-, non-NMDA-, and muscarinic receptor-mediated synaptic transmission, and after 10 min, 3-min samples of the effluent were collected and assayed for [³H]GABA. In the beginning of the ninth sample collection, electrical field stimulation (except where noted, at 50 V, 10 Hz, 3 ms, 360 bipolar square-wave pulses) was applied using a Grass S88 stimulator (Grass Medical Instruments, Quincy, MA) and a pair of platinum ring electrodes.

4

Radioactivity Assay. Previous studies showed that in the presence of aminooxyacetic acid the majority of tritium release under comparable condition represents [³H]GABA (Bernath and Zigmond, 1988). The radioactivity released from the preparations was measured with a Packard 1900 Tricarb liquid scintillation spectrometer (Canberra, Australia). A 0.5-ml aliquot of the perfusate samples was added to 2 ml of scintillation fluid (Packard Ultima Gold) and counts were determined. To determine the residual radioactivity, the tissue was weighed and homogenized, and the radioactivity was extracted with 10% trichloroacetic acid. The release of [³H]GABA was calculated in percentage of the amount of radioactivity in the tissue at the sample collection time (fractional release, %). The tissue tritium uptake was determined as the sum release + the tissue content after the experiment. The release of [³H]GABA evoked by field stimulation (stimulation-evoked release, S) was calculated by the area-under-the-curve (AUC) method, i.e., subtracting the resting release expected to be released, calculated from the pre- and poststimulation period, from the release evoked by simulation, which lasts until a sample tritium content is equal to the prestimulation period sample contents. All data represent the mean ± S.E. of n observations, except EC₅₀ values, which are presented as mean (95% confidence interval) from n experiments. EC₅₀ values were estimated by fitting the data to a sigmoidal logistic equation using the program Prism (GraphPad, San Diego, CA). Statistical significance was calculated by the two-tailed Student's t test, Welch's test, one- or two-way ANOVA followed by Dunnett test, as appropriate, and P < .05 was accepted as significant change.

[Ca²⁺]_i Measurement in Interneurons of Hippocampal Slices. Wistar rats (16-20 days old) were anesthetized (xylazine; Spofa, Prague, Czech Republic plus ketamine; SelBruHa, Budapest, Hungary) and decapitated. The brain was removed and placed in ice-cold cutting solution (composition: 126 mM NaCl, 2.5 mM KCl, 26 mM NaHCO₃, 0.5 mM CaCl₂, 5 mM MgCl₂, 1.25 mM NaH₂PO₄, and 10 mM glucose, pH 7.4 when continuously bubbled with 95% O₂ + 5% CO₂) for 1 to 5 min. Coronal slices (250 µm in thickness) were cut with a vibratome (Vibratome 1000; TPI, St. Louis, MO), separated into left and right halves, and transferred to a mash-bottom holding chamber containing artificial cerebrospinal fluid (ACSF; 126 mM NaCl, 2.5 mM KCl, 26 mM NaHCO₃, 2 mM CaCl₂, 2 mM MgCl₂, 1.25 mM NaH₂PO₄, and 10 mM glucose) bubbled with a mixture of 95% O₂ + 5% CO₂, making the final pH 7.4. After 20- to 25-min incubation at 35.5°C the slices were kept at room temperature until dye loading. For bulk loading of the calcium indicator the slices were incubated in ACSF supplemented with 5 µM fura-2 acetoxymethyl ester (fura-2/AM) (Molecular Probes, Eugene, OR) and 0.025% (w/v) Pluronic F-127 detergent (Molecular Probes) for 45 to 55 min at room temperature. Subsequently the slices were left in ACSF for at least 60 min to ensure fura-2/AM de-esterification. Slices were submerged and superfused (2 ml/min) in an experimental chamber mounted on a Gibraltar BX1 platform (Burleigh, Fishers, NY) and viewed with a 40× water immersion objective in an Olympus BX50WI (Olympus, Hamburg, Germany) microscope. Field stimulation of the slices (10 Hz, 3 ms, 50 pulses) was performed through platinum electrodes placed in two opposite sides of the experimental chamber. Interneurons of the CA1 stratum radiatum and lacunosum moleculare were alternately illuminated at wavelengths 340 ± 5 and 380 ± 5 nm with a TILL Polychrome II monochromator (Planegg, Germany). The UV illumination was attenuated by means of an adjustable diaphragm installed in the light path. The emitted light (510 ± 20 nm) was detected by a cooled charged-coupled device camera (Photometrics Quantix; Photometrics, Tucson, AZ) and the system was controlled by the Axon Imaging Workbench 2.2 software. The [Ca²⁺]_i values of cell somata were calculated off-line using the equation of Grynkiewicz et al. (1985): [Ca²⁺]_i = K_d × F_max³⁸⁰/F_min³⁸⁰ × (R  R_min)/(R_max  R) where R is the ratio of emission intensity, exciting at 340 nm, to emission intensity, exciting at 380 nm; R_min is the ratio at zero free Ca²⁺; R_max is the ratio at saturating Ca²⁺; F_max³⁸⁰ is the fluorescence intensity, exciting at 380 nm, for zero Ca²⁺; and F_min³⁸⁰ is the fluorescence intensity at saturating free Ca²⁺. Intensities of cell images were corrected for the actual background fluorescence obtained from locations close to the fura-2-loaded interneuron. The K_d, F_max³⁸⁰/F_min³⁸⁰, R_min, and R_max values were determined empirically by means of the calcium calibration buffer kit with magnesium No. 2 (Molecular Probes). The parameters K_d, F_max³⁸⁰/F_min³⁸⁰, R_min, and R_max characterizing the system were 182, 8.415, 0.314, and 5.735 nM, respectively.

	Results

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Effect of Nicotinic Receptor Activation on Release of [³H]GABA. Spontaneous [³H]GABA efflux at the beginning of the 15-sample collection period was 0.094 ± 0.008% (n = 16) of the total actual tissue content in the presence of CNQX (10 µM), AP-5 (10 µM), and atropine (10 µM), and remained fairly constant until the end of the experiment. Electrical field stimulation (50 V, 3 ms, 10 Hz, 360 pulses) elicited a rapidly increasing tritium overflow (stimulation-evoked release S = 0.76 ± 0.11%, n = 16), which reached its maximum within 3 min, and declined to the baseline level in the next 6 min upon termination of the stimulus (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effect of nicotine (; n = 16) (100 µM) on the release of [3H]GABA from hippocampal slices. The slices were superfused for 50 min at a rate of 0.6 ml/min with Krebs' solution. After the preperfusion period, glutamate receptor antagonist CNQX (10 µM) and AP-5 (10 µM) and muscarinic receptor antagonist atropine (10 µM) were added to the perfusion solution for 10 min. Subsequently, 3-min samples were collected and assayed for radioactivity. The release of [3H]GABA was expressed as a fractional release (%; see calculation under Materials and Methods). In the 6th min, nicotine perfusion was applied for 30 s, and then the superfusion of the slices continued with nicotine-free medium. Fifteen minutes later, electrical field stimulation was applied (S: 50 V, 10 Hz, 3 ms, 360 pulses). The values show the mean ± S.E. Asterisks represent significant differences from the control (; n = 19) value (***P < .001).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Concentration dependence of the effect of nicotinic receptor agonists on stimulation-induced [3H]GABA release from rat hippocampal slices in the presence of CNQX, AP-5, and atropine. Slices were perfused for 30 s with nicotine in different concentrations ranging from 1 to 200 µM, DMPP (from 30 µM to 1 mM), ACh (from 3 µM to 10 mM), and choline (from 10 µM to 10 mM), respectively, 15 min before the electrical stimulation (50 V, 10 Hz, 360 pulses). The release of [3H]GABA is expressed as fractional release (%) and the evoked release of [3H]GABA was calculated by the AUC method (details under Materials and Methods). The values show the mean ± S.E. of 4 to 19 identical experiments. Asterisks represent significant differences from the control value (*P < .05, **P < .01, ***P < .001). , nicotine (EC50 = 4.1 µM); , choline (EC50 = 352 µM); , ACh (EC50 = 147 µM); , DMPP.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Time dependence of the potentiating effect of nicotine on the evoked release of [3H]GABA in the hippocampal slice. In the presence of CNQX, AP-5, and atropine, the electrical field depolarization (50 V, 10 Hz, 3 ms, 360 pulses) of the slices occurred 15, 30, 45, 60 min, and immediately (0 min) after the 30-s-long perfusion of nicotine (100 µM). The time interval between the perfusion of nicotine and the electrical depolarization of the slices is shown on the x-axis. The release of [3H]GABA is expressed as fractional release (%) and the evoked release of [3H]GABA was calculated by the AUC method (details under Materials and Methods). All values show the mean ± S.E. of 5 to 7 identical experiments. Asterisks display significant differences from the mean of control measured without previous nicotine perfusion (dashed line, ***P < .001).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Voltage and frequency dependence of the effect of nicotine (100 µM) on stimulation-evoked [3H]GABA release from rat hippocampal slices. Slices were exposed to 30-s perfusion of nicotine (100 µM) and 15 min later electrical field stimulation was applied with varying stimulation voltage (25-50 V) or frequency (2-10 Hz) but constant impulse duration (3 ms) and number of shocks (360 pulses). In control experiments slices were stimulated identically, but without previous application of nicotine. CNQX, AP-5, and atropine were present in the perfusion solution in every experiment. The release of [3H]GABA is expressed as fractional release (%) and the evoked release of [3H]GABA was calculated by the AUC method (details under Materials and Methods). Data represent the mean ± S.E. of 6 to 19 identical experiments. Asterisks display significant differences from the mean of control measured without previous nicotine perfusion (*P < .05, ***P < .001).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Inhibition of the effect of nicotine (100 µM) on stimulation-induced [3H]GABA release by nAChR antagonists. Perfusion of each antagonists started 10 min before the sample collection period and continued until the end of the experiment. The experimental protocol was otherwise identical with that shown in Fig. 1.  show the results obtained in experiments without nicotine perfusion;  show the results obtained in experiments when slices were challenged to 30-s perfusion of nicotine. The release of [3H]GABA is expressed as fractional release (%) and the evoked release of [3H]GABA was calculated by the AUC method (details under Materials and Methods). The columns represent the mean ± S.E. of 5 to 19 identical experiments. Asterisks show significant differences from respective controls (*P < .05, **P < .01, ***P < .001).

Effect of Nicotine Perfusion on Evoked Release of [³H]GABA under Ca²⁺-Free Conditions. Because nAChRs are known to be ligand-gated Ca²⁺ channels, it was important to test the Ca²⁺ dependence of the effect of nicotine on the evoked release of [³H]GABA. Perfusion of Ca²⁺-free medium was started 20 min before the sample collection period, and changed to normal Krebs' solution 5 min after the 30-s perfusion of nicotine (100 µM). In Ca²⁺-free medium, the amount of the resting outflow of [³H]GABA was significantly increased (0.39 ± 0.02 and 0.19 ± 0.03%, n = 4, P < .001), but returned to the basal level in the next 12 min after changing to normal Krebs' solution (Fig. 6). When nicotine was perfused in the absence of external Ca²⁺, release of [³H]GABA evoked by subsequent electrical field stimulation, now in the presence of Ca²⁺, was not significantly different from that measured in the absence of nicotine (S = 0.59 ± 0.07 and 0.56 ± 0.02%, n = 4, P > .05; Fig. 6).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   External Ca2+ dependence of the effect of nicotine (; n = 4) (100 µM) on the release of [3H]GABA from hippocampal slices. Perfusion of Ca2+-free medium supplemented with 1 mM EGTA was started 20 min before the sample collection period, and changed to normal Krebs' solution after 5 min of 30-s nicotine (100 µM) perfusion as indicated by horizontal bars. Subsequently, electrical field stimulation was applied (S: 50 V, 10 Hz, 3 ms, 360 pulses). Both solutions were supplemented with CNQX (10 µM) and AP-5 (10 µM), and atropine (10 µM). The values show the mean ± S.E. , Ca2+ free without nicotine, n = 4.

Effect of Nicotine Perfusion on [Ca²⁺]_i in Interneurons in CA1 Hippocampal Region. To examine the effect of nicotine on the level of intracellular Ca²⁺ that can regulate several intracellular signaling pathways, influencing cell function on a longer-lasting time scale (Berridge, 1993) we simulated the conditions in [³H]GABA release experiments and measured [Ca²⁺]_i in the soma of individual interneurons of the CA1 stratum radiatum and lacunosum moleculare layer of hippocampal slices. Perfusion of 100 µM nicotine caused a 108 ± 26 nM increase in the [Ca²⁺]_i (3.1 ± 0.7 times increase compared with the basal [Ca²⁺]_i) in 7 of 20 interneurons (35%). Figure 7A shows a representative experiment. The rest of the interneurons (13) were not responding at all for this type of administration of nicotine. Electrical field stimulation (10 Hz, 3 ms, 50 pulses) after nicotine administration elicited an 85 ± 13 nM [Ca²⁺]_i transient (2.6 ± 0.2 times increase, n = 20), which did not differ significantly from the effect of field stimulation applied in the absence of previous nicotine administration (65 ± 9 nM or 2.9 ± 0.2 times elevation, n = 13, P > .05). When the effect of nicotine was tested in the presence of 10 nM MLA only 2 interneurons (15%) responded by a 37 ± 20 nM increase in [Ca²⁺]_i (1.6 ± 0.03 times enhancement) and 11 remained silent (Fig. 7B). In the presence of 10 µM MEC none of other 10 examined interneurons responded for nicotine.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Representative recordings showing the effect of nicotine perfusion on [Ca2+]i of rat hippocampal CA1 interneurons. A, perfusion of 100 µM nicotine induced a [Ca2+]i transient in an interneuron located at the stratum lacunosum moleculare. B, in the presence of the nicotinic receptor antagonist MLA (10 nM), nicotine failed to enhance [Ca2+]i in 11 of 13 tested interneurons; a CA1 stratum radiatum interneuron. Perfusion of AP-5 (10 µM), CNQX (10 µM), and atropine (0.1-1 µM) started 13 min before and continued until the end of the measurements. Field stimulation (10 Hz, 3 ms, 50 pulses) was delivered through platinum electrodes. Cells were loaded with fura-2/AM and [Ca2+]i of the soma of the interneurons was measured. Light-transmitted DIC images show the respective interneurons the traces were recorded from. Pseudocolor ratio images indicate the [Ca2+]i in the somata of the perfused interneurons at time point before and after nicotine administration.

Effect of PKC and Chloride Channel Inhibition on Nicotine Potentiation of Evoked Release of [³H]GABA. To test the involvement of PKC enzyme in the long-lasting potentiation of [³H]GABA release after short challenge of nicotinic receptor activation, the effect of the nonselective PKC inhibitor staurosporine was also examined. Staurosporine at 100 nM concentration was ineffective to change nicotine-induced potentiation of [³H]GABA release (data not shown). Similarly, flufenamic acid (100 µM) and niflumic acid (100 µM), inhibitors of Cl conductance associated with the activation of 7-type nAChR (Seguela et al., 1993) did not affect increased evoked release in response to nicotine application (data not shown).

Effect of Sodium Channel Inhibition on Nicotine-Induced Potentiation of Evoked Release of [³H]GABA. To examine the sensitivity of the effect of nicotine and the subsequent sensitization of the release apparatus to the Na⁺ channel blocker TTX (1 µM) separately, two different application procedures were used. When TTX was administered 10 min before the sample collection period, and it was redrawn after nicotine application, nicotine (100 µM) was ineffective to potentiate the evoked release of [³H]GABA (S = 0.85 ± 0.14%, n = 4, and S = 0.61 ± 0.18%, n = 4, in the presence and absence of nicotine, respectively; P > .05; Fig. 8). Similar findings were obtained when TTX was introduced after nicotine stimulation and perfused throughout the experiments: no significant potentiation of evoked release of [³H]GABA was observed in response to previous nicotine application (100 µM) under these conditions (S = 0.91 ± 0.46%, n = 6, and S = 0.54 ± 0.21%, n = 6, in the presence and absence of nicotine, respectively; P > .05; Fig. 8).

View larger version (28K): [in this window] [in a new window]   Fig. 8.   Inhibition of sodium channels by TTX (1 µM) prevented the effect of nicotine (100 µM for 30 s) to potentiate the release of [3H]GABA induced by electrical field stimulation (50 V, 10 Hz, 360 pulses). A, TTX perfusion was started 10 min before the sample collection period and finished 12 min before the electrical stimulation. The experimental protocol was otherwise identical with that shown in Fig. 1, i.e., nicotine was administered 15 min before the electrical stimulation, in the presence of TTX. B, TTX was perfusion was started 12 min before the electrical stimulation and continued until the end of the experiment.  show the results obtained in experiments without nicotine perfusion;  show the results obtained in experiments when slices were challenged to 30-s perfusion of nicotine. The release of [3H]GABA is expressed as fractional release (%) and the evoked release of [3H]GABA was calculated by the AUC method (details under Materials and Methods). The columns represent the mean ± S.E. of 4 to 19 identical experiments. Asterisks show significant differences from respective controls (***P < .001).

Role of Membrane Transporters in the Effect of Nicotine. Because it has been shown previously (Nicholls, 1989; Bernath et al., 1993) that intraterminal sodium accumulation, due to strong depolarizing stimuli, may reverse the GABA transporter and elicit GABA release into the extracellular space, the involvement of the GABA transporter in the effect of nicotine was also examined. To separate carrier-mediated and vesicular release of GABA, low temperature and the selective GAT1 GABA transporter inhibitor nipecotic acid (do Nascimento et al., 1998) were tested in our experiments. Cooling the bath temperature to 12-17°C results in the inactivation of integral transporter proteins of cell membrane, a method useful to distinguish between carrier-mediated and exocytotic release of GABA (Vizi, 1998; Vizi and Sperlágh, 1999). Figure 9 demonstrates the results obtained in those experiments that were performed at 12°C. Although the resting outflow of [³H]GABA remained unchanged (0.085 ± 0.0087%, n = 16, and 0.066 ± 0.0023%, n = 4, at 37 and 12°C, respectively; P > .05), the stimulation-evoked [³H]GABA outflow was approximately 1.7 times higher at 12°C than at 37°C (Fig. 9), which might be due to the blockade of the inward transport of the released GABA. At low temperature, nicotine (100 µM) was completely ineffective to increase stimulation-evoked [³H]GABA outflow (Fig. 9).

View larger version (31K): [in this window] [in a new window]   Fig. 9.   Low temperature (12°C) or nipecotic acid (1 mM), both inhibits the GABA transport system, fully abolished the effect of nicotine (100 µM) on electrical stimulation-induced [3H]GABA release. Nipecotic acid perfusion was started 10 min before the sample collection period and continued until the end of experiment. The experimental protocol was otherwise identical with that shown in Fig. 1.  show the results obtained in experiments without nicotine perfusion;  show the results obtained in experiments when slices were challenged to 30-s perfusion of nicotine. The release of [3H]GABA is expressed as fractional release (%) and the evoked release of [3H]GABA was calculated by the AUC method (details under Materials and Methods). The values represent the mean ± S.E. of 4 to 19 identical experiments. Asterisks show significant differences from respective control (***P < .001) or from 37°C control (*P < .05), as indicated.

	Discussion

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In this study we explored the effect of the activation of nAChRs located on hippocampal GABAergic interneurons by 1) measuring GABA release from superfused hippocampal slices at rest and in response to depolarizing stimuli, and 2) monitoring [Ca²⁺]_i in individual hippocampal interneurons.

Modulation of GABA Release by Nicotinic Agonists. Recent electrophysiological findings indicated that functional 42-like and 7-type nicotinic ACh receptors are present on subsets of hippocampal interneurons (Alkondon et al., 1997, 1998; Frazier et al., 1998a,b; McQuiston and Madison, 1999). Nevertheless, application of nicotinic agonists, i.e., nicotine, ACh, and choline failed to affect basal [³H]GABA outflow in our experiments, which could be explained by 1) only a subpopulation of interneurons express nAChR, and in our model, where the outflow of GABA from the whole slice is measured, no change in the overall [³H]GABA outflow is detectable; 2) because a number of studies showed that subthreshold doses of nicotine readily desensitize nicotinic receptors (Frazier et al., 1998; Lu et al., 1999) it might occur under our experimental conditions that desensitization of nAChR is gradually developed by subactivating concentrations of nicotine reaching the perfusion chamber earlier than the bulk of nicotine-containing solution; or 3) by the simple activation of somatodendritic or preterminal nAChR by nicotinic agonists is ineffective to elicit repetitive action potentials spreading to axon terminals and give rise to detectable GABA release; instead, subsequent depolarization is necessary to amplify the signal of nicotinic receptor activation to result in a measurable amount of GABA release. Indeed, when the interneurons were depolarized synchronously by electrical field stimulation at 10 Hz, a strongly increased [³H]GABA outflow was observed after perfusion of slices by nicotinic receptor agonists nicotine, choline, and ACh. Because the excitatory transmission was blocked by CNQX, AP-5, and atropine, it is unlikely that nicotine acted indirectly upon GABAergic cells by facilitating excitatory neurotransmission during the electrical field stimulation. In addition, it can also be exluded that disinhibiton of GABAergic cells was the reason for the increased [³H]GABA release because activation of nicotinic receptors is known to excite the neurons (Gray et al., 1996; Jones and Yakel, 1997; Albuquerque et al., 1998; McQuiston and Madison, 1999). Thus, it is concluded that the direct activation of interneuronal nAChR by nicotine treatment was responsible for the potentiation of electrical depolarization-dependent [³H]GABA outflow. The effect of nicotine, ACh, and choline depended on their concentration; however, nicotine, above 100 µM, and ACh, above 3 mM, did not enhance the evoked [³H]GABA release significantly, which may result from uncoupling of intracellular signaling machineries to supramaximal activation of nAChR or recruitment of other nAChRs at these concentration that directly or indirectly inhibits GABA release. Another nicotinic agonist DMPP was ineffective to induce significant potentiation of evoked [³H]GABA release, indicating that it is a weak agonist at this receptor, similarly to chick 7 expressed in Xenopus oocytes (Bertrand et al., 1992). Moreover, DMPP shows anomalous behavior consistent with channel block at rat 7 (Seguela et al., 1993) and exhibits Ca²⁺-independent and mecamylamine-insensitive action when tested on striatal [³H]dopamine efflux (El-Bizri and Clarke, 1994; Clarke and Reuben, 1996).

Nicotinic Receptor Subunits Participating in the Modulation of Evoked [³H]GABA Release. In this study we have described the effect of different nicotinic receptor agonists on electrically evoked [³H]GABA release, and the sensitivity of nicotine-enhanced evoked [³H]GABA release to the blockade by a variety of nAChR antagonists. The reversal of the effect of nicotine by -BTX or MLA, which are 7 subtype-selective antagonists in nanomolar range (Alkondon et al., 1992; Clarke and Reuben, 1996), and the effectiveness of the 7 subtype-selective agonist choline to elicit a similar potentiation (Alkondon et al., 1999) strongly suggest the participation of 7 subunit-bearing nAChR in the effect of nicotine. In contrast, DHE at nanomolar concentration, at which it antagonizes 42 receptors mediated currents in culture (Alkondon and Albuquerque, 1993) and in slice (Alkondon et al., 1999), had no effect. Although DHE, when applied at high concentration, and MEC, the nonselective nAChR antagonist, also prevented the action of nicotine, which would be suggestive for the involvement of other subunit assemblies, these findings rather indicate that DHE and MEC have activity on hippocampal 7-type nAChR. Indeed, 7 receptor-mediated type IA currents have been also shown to be sensitive to DHE in the micromolar range in hippocampal neurons (Alkondon and Albuquerque, 1993), slice (Alkondon et al., 1999), and Xenopus oocyte (Bertrand et al., 1992). Similarly, ACh-evoked, 7-mediated currents recorded from hippocampal neurons (Alkondon and Albuquerque, 1993), and from interneurons in the hippocampal slice (Jones and Yakel, 1997), were inhibited by MEC in concentrations that used in our study. Furthermore, GABA release initiated by 42-like receptor activation proved to be -BTX insensitive and only partly TTX sensitive in hippocampal synaptosomes (Lu et al., 1998), whereas both -BTX and TTX completely abolished potentiation of [³H]GABA release by nicotine in the present study. These findings suggest that the receptor population responsible for the two effects is different, and the involvement of the 42 subtype in this particular effect of nicotine does not appear likely.

Mechanism Underlying the Facilitation of [³H]GABA Release by Nicotine. Neuronal nAChR are ligand-gated ion channels that are more permeable to Ca²⁺ than their neuromuscular nAChR counterpart (Seguela et al., 1993). ACh, by opening the ion channel of its receptors and causing a brief inward Ca²⁺ and Na⁺ current, can elicit action potentials, which may result in subsequent Ca²⁺ influxes via voltage-dependent Ca²⁺ channels (Barrantes et al., 1995), and may also regulate target cell function via Ca²⁺ level-dependent processes in a longer time scale (in the range of minutes) (Berridge, 1993).