Abstract

Nicotine stimulation of cortical neurons obtained from gestation day 19 rats provoked a dose-dependent release of aspartate, glutamate, glycine and GABA, indicating a functional role for the nicotinic receptor in this model. This release was exclusively Ca2+-dependent (vesicular release) in the case of aspartate and dual (Ca2+-dependent and Ca2+-independent) for glutamate, glycine and GABA. Nicotine also raised the membrane potential and the intracellular calcium concentration. These effects were specific, since they were reversed by hexamethonium, an antagonist of the nicotinic receptor. It was shown that L, N, and P/Q type Ca2+ channels are involved in nicotine-mediated Ca2+ entry into cortical neurons. Evaluation of the effects of nicotine on Ca2+ entry in isolated cells showed that 100% of the cells responded to nicotine, although the intensity of the response was variable: 63% of the neurons showed an increase in intracellular Ca2+ of 152 ± 5 grey levels, 25% of 88 ± 12 grey levels and 12% of 48 ± 1 grey levels. Tetrodotoxin, which blocks voltage-dependent Na+ channels, completely reversed nicotine-induced Ca2+ entry into single cells. This suggests that the Ca2+ increment is mediated by opening of Ca2+ channels and not by the nicotinic receptor.

Introduction

Although its physiological and psychological effects have long suggested that nicotine exerts specific actions in the brain, the identification of neuronal nicotinic receptors has only been possible in the last few years due to the development of molecular biology techniques [reviewed by McGehee and Rolen (McGehee and Rolen, 1995)]. A variety of nicotinic acetylcholine receptor (nAchR) complexes in the central nervous system (CNS) are formed by a diverse array of subunits which confer different pharmacological and physiological properties to the receptors (Sargent, 1993; Rust et al., 1994). Although there is little experimental evidence (Anano et al., 1991; Cooper et al., 1991), the neuronal nAchR channel is assumed to be a pentamer formed by α and β subunits, although many alternative α/β stoichiometries may exist, and this could contribute to the particular functionality of the nAchR channel. The expression of specific nAchR subunits is important during brain development since it influences neuronal excitability (Margiotta and Gurantz, 1989). Nicotinic AchRs are present on autonomic neurons and adrenal chromaffin cells of the peripheral nervous system and on many neurons in the CNS. Many of the properties of the nAchR channels, such as ion selectivity and gating properties, resemble those of muscle AchR. However, neuronal AchRs are clearly distinct from muscle and are themselves diverse. The most important role performed by cholinergic receptors in the CNS is their participation in neuronal excitability modulation (Aquilonius and Gillberg, 1990; Vernino et al., 1992) and in neurotoxicity events (Slotkin et al., 1997). Cumulative evidence from animal and human studies has indicated that nicotinic systems play a major role in higher cognitive functions and dysfunctions. Nakayama et al. were able to show the presence of nicotinic receptors in cerebral cortex through immunocyto-chemical techniques (Nakayama et al., 1995). The number of these receptors seems to be diminished in neurological disorders such as Parkinson's and Alzheimer's diseases (Araujo et al., 1988; Schroder et al., 1995; Chesselli, 1997; Zamanim et al., 1997) and schizophrenia (Miller et al., 1996). Pharmacological and electrophysiological studies appear to suggest the existence of different isoforms of the nicotinic receptor in the neocortex and hippocampus (Wada et al., 1989, 1990; Seguela et al., 1993; Lobron et al., 1995), although the exact location of these isoforms is not known. This diversity of nicotinic receptors in the CNS makes functional and pharmacological variations possible (Papke et al., 1989; Luetje and Patrick, 1991). In dopaminergic nigrostriatal axons nicotine induces dopamine release after previous stimulation of glutamatergic neurons, which release glutamate (García Muñoz et al., 1996). In hippocampal synaptosomes release of noradrenaline may be modulated by nicotinic receptors containing α3 and β4 subunits (Clarke and Reuben, 1996). Furthermore, nicotine may induce increases in glutamate, dopamine, serotonin and acetylcholine release in the hippocampus, cerebellum, nucleous accumbens and striatum (Thoth et al., 1992; Marshall et al., 1996, 1997; Wilkie et al., 1996; Ferger and Kuschinsky, 1997). Conversely, Izenwasser et al. found that in the striatum nicotine inhibits [3H]dopamine uptake and induces its release (Izenwasser et al., 1991). Moreover, Waniewski and Martin reported that the stimulation of sympathetic ganglia with nicotinic or muscarinic agonists provoked the release of [3H]taurine (Waniewski and Martin, 1994). Although there is much data on the properties and functional characteristics of the different cholinergic receptors (AchR) in the CNS, most of these may be considered presynaptic receptors since the action of nicotine has generally been evaluated in terminal neurons (synaptosomes). However, nicotinic receptors are distributed throughout the neurons. Few investigations have centred on the action of AchRs on neuro-transmitter release in neuronal preparations, although the effects of nicotine in in vivo systems have also been studied (Thoth et al., 1993). The aim of the present study was to investigate the nicotinic receptor-mediated modulation of synaptic transmission in cortical neurons. Our results suggest that in cortical neurons in culture nicotine induces aspartate release by an exocytotic pathway and glutamate, glycine and GABA release by a dual mechanism. It was also established that nicotine increased the intracellular calcium concentration in all of the neurons examined.

Materials and Methods

Materials

Fura 2AM and bis-[1,3-diethylthiobarbiturate]trimethine oxonol (bis-oxonol) were obtained from Molecular Probes (Eugene, OR). Eagle's minimum essential medium (EMEM) was supplied by Bio-Whittaker and foetal calf serum (FCS) and horse serum (HS) by Sera-Lab (Sussex, UK). Nicotine, hexamethonium and muscimol came from Sigma (St Louis, MA). The remaining chemicals were reactive grade products from Merck (Darmstadt, Germany).

Cell Isolation and Culture

Brain neurons were obtained from foetal rat brains at gestation day 19 following the procedure described by Segal (Segal, 1983) with minor modifications. Isolated neurons were suspended in EMEM containing 0.3 g/l glutamine, 0.6% glucose, 10% FCS, 10% HS, 100 U/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamycin and 5 μg/ml imipenem. Cells, at a density of 2 × 106 cells/well, were plated onto plastic Petri dishes treated with 10 μg/ml poly-l-lysine to aid attachment. The plates were incubated in a humidified incubator in an atmosphere of 5% CO2/95% air at 37°C. After 72 h the incubation medium was replaced with fresh medium to which 10 μM cytosine arabinoside was added to prevent overgrowth of contaminating glial cells. Cells were used after 10–15 days culture. Cell viability was checked by the trypan blue exclusion method. Viability was routinely >95%. Cell purity was checked by both cell staining with cresyl violet to identify neurons and with a specific anti-GFAP antibody to identify glial cells.

Glial Contamination

After 10–15 days culture the cortical neurons were detached from the culture plates with trypsin, as indicated below. Cells were the fixed (for 30 min) in 2% paraformaldehyde and washed in phosphate-buffered saline (PBS) followed by treatment (1 h) with anti-rabbit GFAP antibody (diluted 1/500). Cells were once again washed in PBS and treated with anti-rabbit FITC-conjugated IgG at a dilution of 1/100 for 30 min and identified by flow cytometry. Under these conditions the glial cells in the cultures were estimated at 9 ± 3% of the total cell population (neural + glial cells).

Cytosolic [Ca2+]

Changes in intracellular calcium concentration, [Ca2+]i, were monitored by Fura 2AM fluorescence. Six day cultured cells were detached from the plates using trypsin (0.25% trypsin and 0.02% EDTA in Dulbecco's phosphate-buffered saline without calcium or magnesium) and washed twice using 1 ml of a Krebs HEPES solution (Locke medium) containing 140 mM NaCl, 4.4 mM KCl, 2.5 mM CaCl2, 1.2 mM Mg(SO4)2, 1.2 mM KH2PO4, 4.0 mM NaHCO3, 5.5 mM glucose, 0.58 mM ascorbic acid and 10 mM HEPES, adjusted to pH 7.5 and incubated with 5 μM Fura 2AM for 45 min at 37°C. Excessive dye was removed by washing the cells twice with fresh Locke medium followed by suspension in this medium at 1 × 106 cells/ml. After Fura 2AM treatment, cell viability was checked as indicated previously. Fluorescence (excitation wavelength 340/380 nm, emission 510 nm) was monitored at 37°C in a well stirred cuvette containing 1 ml of this suspension using a Perkin Elmer LS-50 spectro-fluorimeter (slits 5 nm excitation, 10 nm emission). At the end of each experiment 1% Triton X-100 was added to make the cells permeable and permit the dye to gain access to the extracellular Ca2+ (2.5 mM). This Ca2+ concentration saturated the dye and provided a measure of the maximum fluorescence signal (Fmax). To determine the minimum fluorescence signal (Fmin), 20 mM Tris base was added to raise the pH above 8.2, followed by 5 mM EGTA which reduced the Ca2+ to <1 nM. [Ca2+]i was calculated with the Grynkiewicz equation (Grynkiewicz et al., 1985):

 

\[[\mathrm{Ca}^{2{+}}]_{i}\ {=}\ \mathit{K}_{\mathrm{d}}\ [(\mathit{F}\ {\mbox{--}}\ \mathit{F}_{\mathrm{min}})/(\mathit{F}_{\mathrm{max}}\ {\mbox{--}}\ \mathit{F})]\ {\times}\ (\mathrm{SF}_{2}/\mathrm{SB}_{2})\]

where F is the ratio between fluorescence values at 340 and 380 nm, SF2 is the maximum fluorescence at 380 nm and SB2 is the minimum fluorescence at 380 nm. The equilibrium dissociation constant (Kd) for the complex [Ca2+]–Fura 2AM was of 224 nM.

Single Cell [Ca2+]i

Cells cultured on glass coverslips were loaded for 45 min with Fura 2AM (5 μM) dissolved in Locke medium. After incubation at 37°C in the dark, cells were washed with Locke medium and the coverlips placed under a Nikon inverted stage microscope under continuous perfusion with Locke medium. Light from a xenon lamp was filtered through two different band-pass filters (340 or 380 nm) in the excitation path and the specimen illuminated on the microscope stage by a dichroic mirror. Excitation wavelengths of 340 and 380 nm were alternately applied to the cells. The fluorescence emitted by the cells was passed through a band-pass filter (510 nm) and video images were obtained using a cold intensified camera. Ratios corresponding to 340/380 nm are given as grey levels. Output from the camera was digitalized and stored in a computerized imaging system (MiraCal). The reagents dissolved in Locke medium were applied in the perfusion medium. When tetrodotoxin (TTX) was used the order of nicotine and TTX applications was as follow. First neurons were stimulated with 10 μM nicotine and images were taken over 80 s. Then, cells were perfused for 10 min to permit recovery of the nicotinic receptor. Subsequently, neurons were stimulated with 10 μM nicotine plus 50 nM TTX and images taken for 80 s as before.

Membrane potential

Changes in the membrane potential of neurons were monitored using the fluorescent dye bisoxonol. This is a lipophilic anion whose distribution across the membrane is dependent upon the membrane potential. Thus, an increase in bisoxonol fluorescence indicates that the membrane has been depolarized, allowing more of this negatively charged dye to enter the cells (Waggoner, 1979). Washed cells, as used for [Ca2+]i determination, were suspended in Locke medium at a density of 5 × 105 cells. Neurons in suspension were incubated with 0.2 μM bisoxonol for 10–20 min and placed in a fluorimeter. Fluorescence was measured at an excitation wavelength of 540 nm and emission wavelength of 565 nm and monitored at 37°C in a well stirred cuvette using a Perkin Elmer spectrofluorimeter. Drugs were added at the indicated concentrations. Controls were performed using Locke medium in place of the drug. Fluorescence intensity is reported in arbitrary units.

Amino Acid Secretion

High performance liquid chromatography (HPLC) of amino acids was performed according to the methods described by Márquez et al. (Márquez et al., 1986). Cells were washed twice at 10 min intervals with 1 ml of Locke medium. After removal of the medium cells were stimulated for 15 min periods at 37°C with 0.5 ml of fresh Locke medium containing the different secretagogues. The stimulating medium was then withdrawn and cells ruptured by the addition of 0.5 ml of distilled water. The concentration of amino acids was determined by reversed phase HPLC using pre-column derivation with dansyl chloride and UV detection at 254 nm. Integration of peaks was achieved using a Sprectraphysis integrator. Peaks were quantified by comparison with those obtained using simultaneously prepared amino acid standards. Separation of dansyl derivatives was performed using a 5 μM Spherisorb-ODS-2 column (15 × 0.46 cm).

Proteins were identified according to Bradford (Bradford, 1976). Results were expressed as nmol neurotransmitter/mg protein/well or as the percentage of amino acids released into the incubation medium with respect to the total amino acid content (incubation medium + cells).

Data Presentation

Data are presented as the means of three or four separate experiments performed on different cell cultures. Each experiment was performed in duplicate using different batches of cells. Student's t-test was used to statistically compare data.

Results

A dose-dependent release of the amino acids aspartate, glut-amate, glycine and GABA was shown when cortical neurons were stimulated with nicotine. The effect of nicotine was first observed at a nicotine dose of 50 μM. Glycine was released in greatest quantity (Fig. 1). Measurement of secretion mediated by nicotine in a medium with and without external calcium revealed that aspartate release was exclusively Ca2+-dependent (vesicular release), while the release of the remaining amino acids occurred through Ca2+-dependent and Ca2+-independent processes. The order of Ca2+-dependent release of these amino acids at a nicotine concentration of 200 μM was: aspartate ≃ glycine > glutamate > GABA. The Ca2+-dependent release of all of these amino acids was higher than that observed in calcium-free medium, except in the case of GABA, which was preferentially released by a Ca2+-independent process (Fig. 2).

The nicotinic acetylcholine receptor antagonist hexamethonium completely blocked the release of these amino acid neuro-transmitters evoked by nicotine, which implies that the effect may be attributed to activation of the nicotinic receptor (Fig. 3).

Nicotine also increased both the membrane potential, measured as arbitrary fluorescence units, and intracellular calcium levels in a dose-dependent manner. Both effects were inhibited by hexamethonium (Fig. 4A,B).

Release of the four amino acid neurotransmitters evoked by 200 μM nicotine was inhibited by verapamil, an antagonist of L type Ca2+ channels; ω-conotoxin GVIA, an antagonist of N type Ca2+ channels, inhibited the release of all the amino acid neuro-transmitters with the exception of aspartate. Further, ω-agatoxin GIVA, which blocks P/Q type Ca2+ channels, inhibited the release of aspartate, glycine and GABA, but not of glutamate (Fig. 5).

When the nicotine-mediated increments in intracellular calcium levels were measured in single cells it was observed that all of the neurons responded to nicotine, although these responses varied depending on the cell. Increases in intracellular Ca2+ levels of 152 ± 5 grey levels were recorded in 63% of the cortical neurons, of 90 ± 10 in 25% and of 48 ± 1 in 12% (Fig. 6).

TTX, which blocks voltage-dependent Na+ channels, completely reversed the Ca2+ entry mediated by nicotine in 100% of the cells under study (Fig. 7).

Discussion

Despite abundant data on AchR diversity in the CNS, there is still little evidence for classical nicotinic synaptic transmission. The results presented here clearly demonstrate that when cortical neurons are stimulated with concentrations of nicotine from 50 to 200 μM there is a dose-dependent release of aspartate, glutamate, glycine and GABA, which is exclusively Ca2+-dependent (exocytotic release) or dual (Ca2+-dependent and Ca2+-independent), depending on the amino acid. The nicotine concentration needed to induce amino acid release appears high compared with the findings of electrophysiological studies (McGehee et al., 1995). However, it is much lower than the concentrations used (1 mM or higher) in studies in which amino acid release was measured directly (Thoth et al., 1993). Besides its effects on amino acid neurotransmitter release, nicotine also induced membrane depolarization and intracellular Ca2+ increases. All these effects were blocked by hexamethonium, an antagonist of the nicotinic receptor, indicating their specificity. Intracellular calcium increases mediated by this cholinergic receptor were also shown by Mulle et al. in rat CNS neurons (Mulle et al., 1992). Further, Letz et al. observed that the stimulation of cholinergic receptors on pinealocytes may cause membrane depolarization and activation of L type Ca2+ channels (Letz et al., 1997).

The data presented not only demonstrate the presence of nicotinic receptors in cultured cortical neurons, but also indicate their specificity and functionality. These findings are in accordance with those obtained by Vidal and Changeus, who reported an increase in the negative wave of field potentials reflecting increased excitability of cortical neurons when neocortical slices were treated with acetylcholine or dimethylphenyl piperazinium (Vidal and Changeus, 1989).

In the present study nicotine was also able to induce the release of amino acid neurotransmitters in a calcium-free medium (Ca2+-independent release). This Ca2+-independent amino acid release may be attributable to the reverse action of amino acid transporters, given that the nicotinic receptor is a Na+-permeable channel and, when open, the resulting increased intracellular Na+ level is a condition required to activate amino acid transporters.

Our data seem to indicate that different Ca2+ channels might be involved in release of the different amino acid neurotrans-mitters evoked by nicotine, since blockers of L, N and P/Q type Ca2+ channels inhibited release of the inhibitory amino acid neurotransmitters (glycine and GABA). However, N type Ca2+ channel blockers did not affect aspartate release and P/Q type Ca2+ channel blockers did not affect release of glutamate. These data would appear to suggest that opening of one Ca2+ channel or another is important in secretion of the different amino acid neurotransmitters mediated by nicotine.

The fact that nicotine-stimulated cortical neurons release excitatory (aspartate and glutamate) and inhibitory (glycine and GABA) amino acids would seem to indicate that nicotinic receptors are able to modulate excitatory and inhibitory synapses in cortical neurons.

An observation that warrants particular attention was the high release of aspartate produced when cortical neurons were stimulated with high concentrations of nicotine. This might indicate that in this part of the brain the toxic effect attributed to overexcitability produced by nicotine may be due not only to glutamate release but also to aspartate release that, like glutamate, is able to bind to NMDA receptors. The high release of glycine mediated by nicotine is also of note. Glycine release was some four times higher than GABA release. As both these amino acids are inhibitory, it may be considered that in nicotine-stimulated cortical neurons glycine may mediate inhibition or modulate the response of the NMDA receptor. This matter requires further study.

It may be inferred from the study of Ca2+ entry mediated by nicotine in single neurons that most cortical neurons have nicotinic receptors, although the possibility that Ca2+ entry could be mediated by excitatory amino acids released by nicotine should not be discarded. The increment in intracellular Ca2+ mediated by nicotine could be due to Ca2+ entry through: (i) voltage-dependent Ca2+ channels, since nicotine induced membrane depolarization, and/or (ii) Ca2+ entry through the nicotinic receptors, since, according to some authors, neuronal AchRs show significant permeability to Ca2+ (Adams and Nulter, 1992; Vernino et al., 1992, 1994). This Ca2+ permeation is sufficient to activate Ca2+-dependent cellular processes. However, in the presence of TTX, which completely blocked the Ca2+ entry mediated by 10 μM nicotine, the intracellular Ca2+ increment evoked by nicotine appeared to be mediated by the opening of Ca2+ channels and not by the nicotinic receptor. In this case the nicotinic receptors in cortical neurons seems to be impermeable to Ca2+ ions. These results agree with those of Lena and Changeux, which showed that the depolarizing effect of nicotine in thalamic neurons appears to be mediated through entry of calcium through voltage-sensitive calcium channels (Lena and Changeux, 1997). This is not surprising given the two major functional properties of neuronal nAChRs established by Vernino et al. (Vernino et al., 1992). These authors described nAChRs which show substantial permeability to Ca2+ and a further population of receptors which do not appear to permit Ca2+ movement. These differences may be attributed to the high degree of heterogeneity of neuronal nAChRs brought about by the combination of different subunits to give rise to many structural and functional variants of this neuronal receptor (Boulter et al., 1987; Duvoisin et al., 1989; Couturier et al., 1990).

Herrero et al. recently demonstrated that the Ca2+ channel blockers used in the present study also block α7 and α3β4 AchRs in chromaffin cells (Herrero et al., 1999), giving rise to the possibility that the present effect could be due to blockade of the nAchR. However, our results indicate that the effect of the toxins is mediated by Ca2+ channel blockade given that (i) the toxins show different sensitivity in inhibiting release of the different amino acid neurotransmitters and (ii) TTX, a voltage dependent Na+ channel blocker, completely abolished the Ca2+ entry mediated by 10 μM nicotine.

It may be concluded that: (i) cortical neurons contain functional nicotinic receptors since when stimulated with nicotine these neurons release aspartate, glutamate, glycine and GABA; (ii) the mechanism by which nicotine induces amino acid release is exocytotic or dual, depending on the amino acid; (iii) the effect of nicotine is specific since it is blocked by hexamethonium; (iv) L, N and P/Q type Ca2+ channels are involved in the nicotine effect.

Notes

This work was supported by CAICYT grants PM98-0121 and CAM 08.8/0012/1998. E.L. is the recipient of fellowships from the Ministerio de Educación y Ciencia. S.V. is the recipient of a fellowship from UC. We thank M. García Mauriño for helping us with culture preparation.

Address correspondence to M.P. González, Instituto de Bioquímica (Centro Mixto CSIC-UCM), Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain. Email: pilarg@eucmax.sim.ucm.es

Figure 1.

Effect of different nicotine concentrations on amino acid neurotransmitter secretion in cortical neurons in culture. Basal is spontaneous release in the absence of nicotine, results without stimulation. Results are given as means ± SEM of two separate experiments, from cells of different cultures, each one performed in triplicate. Statistical significance is with respect to basal amino acids release. NS, not significant; ***, P < 0.001.

Figure 1.

Effect of different nicotine concentrations on amino acid neurotransmitter secretion in cortical neurons in culture. Basal is spontaneous release in the absence of nicotine, results without stimulation. Results are given as means ± SEM of two separate experiments, from cells of different cultures, each one performed in triplicate. Statistical significance is with respect to basal amino acids release. NS, not significant; ***, P < 0.001.

Figure 2.

Effect of 200 μM nicotine on amino acid neurotransmitter release measured in a medium with (total release) and without external Ca2+ (Ca2+-independent release). Ca2+-dependent amino acid release is the difference between total release and release found in the absence of calcium in the medium. All results are given after subtracting the correspondent basal values. *, statistical significance with respect to the corresponding basal values (with and without external calcium). •, statistical significance between release measured in the presence and absence of external calcium. •• or **, P < 0.01, ••• or ***, P < 0.001.

Figure 2.

Effect of 200 μM nicotine on amino acid neurotransmitter release measured in a medium with (total release) and without external Ca2+ (Ca2+-independent release). Ca2+-dependent amino acid release is the difference between total release and release found in the absence of calcium in the medium. All results are given after subtracting the correspondent basal values. *, statistical significance with respect to the corresponding basal values (with and without external calcium). •, statistical significance between release measured in the presence and absence of external calcium. •• or **, P < 0.01, ••• or ***, P < 0.001.

Figure 3.

Effect of 200 μM hexamethonium on the amino acid release evoked by 50 μM nicotine. Results are means of two separate experiments, with different cultures, each one performed in duplicate. Values are given after subtracting the basal values. ***, P < 0.001.

Figure 3.

Effect of 200 μM hexamethonium on the amino acid release evoked by 50 μM nicotine. Results are means of two separate experiments, with different cultures, each one performed in duplicate. Values are given after subtracting the basal values. ***, P < 0.001.

Figure 4.

Effect of nicotine on: (A) membrane potential, measured as arbitrary fluorescence units (as indicated in Materials and Methods), in the presence and absence of 200 μM hexamethonium; (B) increase in intracellular Ca2+, measured in the absence and presence of 200 μM hexamethonium. The basal intracellular calcium concentration was 141 ± 20 nM. Results are means of two or three separate experiments, with cells of different cultures, each one performed in duplicate or triplicate.

Figure 4.

Effect of nicotine on: (A) membrane potential, measured as arbitrary fluorescence units (as indicated in Materials and Methods), in the presence and absence of 200 μM hexamethonium; (B) increase in intracellular Ca2+, measured in the absence and presence of 200 μM hexamethonium. The basal intracellular calcium concentration was 141 ± 20 nM. Results are means of two or three separate experiments, with cells of different cultures, each one performed in duplicate or triplicate.

Figure 5.

Effect of calcium channel antagonists on amino acid neurotransmitter release evoked by 200 μM nicotine. *, statistical significance between amino acid release evoked by 200 μM nicotine in the absence and presence of calcium channel antagonists. Results are means ± SEM of two separate experiments from different cultured cells, each one performed in duplicate. Statistical significances are given between intracellular Ca2+ increments induced by nicotine or nicotine with the corresponding Ca2+ channel antagonist. NS, not significant; *, P > 0.05; ***, P > 0.001.

Figure 5.

Effect of calcium channel antagonists on amino acid neurotransmitter release evoked by 200 μM nicotine. *, statistical significance between amino acid release evoked by 200 μM nicotine in the absence and presence of calcium channel antagonists. Results are means ± SEM of two separate experiments from different cultured cells, each one performed in duplicate. Statistical significances are given between intracellular Ca2+ increments induced by nicotine or nicotine with the corresponding Ca2+ channel antagonist. NS, not significant; *, P > 0.05; ***, P > 0.001.

Figure 6.

Whole neuron recordings of intracellular calcium increments evoked by 10 μM nicotine in single cells. Traces show representative examples of typical F340/F380 ratio responses (grey levels) produced in a single neuron after stimulation with the indicated agent. Measurements were performed in medium with external calcium. The insert indicates the percentage of cells with similar behaviour. The number of neurons analysed was 35 from three different cultures.

Figure 6.

Whole neuron recordings of intracellular calcium increments evoked by 10 μM nicotine in single cells. Traces show representative examples of typical F340/F380 ratio responses (grey levels) produced in a single neuron after stimulation with the indicated agent. Measurements were performed in medium with external calcium. The insert indicates the percentage of cells with similar behaviour. The number of neurons analysed was 35 from three different cultures.

Figure 7.

Whole neuron recording of intracellular calcium increments evoked by 10 μM nicotine in the absence or presence of 1 μM tetrodotoxin. Traces are representative examples of typical F340/F380 ratio responses, expressed as grey levels, produced in a single neuron after stimulation with the indicated agents, as indicated in Materials and Methods. Measurements were performed in medium with external calcium. The number of neurons analysed was 15 from two different cultures.

Figure 7.

Whole neuron recording of intracellular calcium increments evoked by 10 μM nicotine in the absence or presence of 1 μM tetrodotoxin. Traces are representative examples of typical F340/F380 ratio responses, expressed as grey levels, produced in a single neuron after stimulation with the indicated agents, as indicated in Materials and Methods. Measurements were performed in medium with external calcium. The number of neurons analysed was 15 from two different cultures.

References

Adams DJ, Nulter TJ (
1992
) Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.
J Physiol (Paris)
 
85
:
67
–76.
Anano R, Conroy WG, Schoepfer R, Whithin GP, Lindstrom J (
1991
) Neuronal nicotinic acetylcholine receptors expresed in xenopus oocytes have a pentameric quaternary structure
J Biol Chem
 
266
:
11192
–11198.
Aquilonius SM, Gillberg PG (1990) Cholinergic neurotransmission: functional and clinical aspects. Amsterdam: Elsevier.
Araujo DH, Lapchak PA, Robitaille Y, Gauthier S, Quirion R (
1988
) Differential alteration of various cholinergic markers in cortical and subcortical regions of human brain in Alzheimer's disease.
J Neurochem
 
50
:
1914
–1923.
Boulter J, Connolly J, Deneris E, Goldman D, Heinemann S, Patrick J (
1987
) Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family.
Proc Natl Acad Sci USA
 
84
:
7763
–7767.
Bradford M (
1976
) A rapid, sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
 
72
:
248
–253.
Chesselll JP (
1997
) Acetylcholine receptor targets on cortical pyramidal neurones as targets for Alzheimer's therapy.
Neurodegeneration
 
5
:
453
–459.
Clarke PB, Reuben M (
1996
) Release of [3H]-noradrenaline from rat hippocampal synaptosomes by nicotine modulation by different nicotinic receptors from striatal [3H]-dopamine release.
Br J Pharmacol
 
117
:
595
–606.
Cooper E, Couturier S, Ballivet M (
1991
) Pentameric structure and subunit stoichometry of a neuronal acetylcholine receptor.
Nature
 
350
:
235
–238.
Couturier S, Bertrand D, Matter JM, Hernandez MC, Bertrand S, Millar N, Valera S, Barkas T, Ballivet M (
1990
) A neuronal nicotinic acetyl-choline receptor subunit (α7) is developmentally regulated and forms a homo-oligomeric channel blocked by α-BTX.
Neuron
 
5
:
847
–856.
Duvoisin RM, Deneris ES, Patrick J, Heinemann S (
1989
) The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit; β4.
Neuron
 
3
:
487
–496.
Ferger B, Kuschinsky K (
1997
) Biochemical studies support the assumption that dopamine plays a minor role in the EEG effects of nicotine.
Phychopharmacology (Berl)
 
129
:
192
–196.
Garcia-Muñoz M, Patino P, Young SJ, Groves PM (
1996
) Effects of nicotine on dopaminergic nigrostriatal axons requires stimulation of pre-synaptic glutamatergic receptors.
J Pharmacol Exp Ther
 
277
:
1685
–1693.
Grynkiewicz G, Poenie M, Tsien RY (
1985
) A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
 
260
:
3440
–3450.
Herrero CJ, Garcia-Palomero E, Pintado AJ, Garcia AG, Montiel C. (
1999
) Differential blockade of rat alpha3beta4 and alpha7 neuronal nicotinic receptors by omega-conotoxin MVIIC, omega-conotoxin GVIA and diltiazem.
Br J Pharmacol
 
127
:
1375
–1387.
Izenwasser S, Jacobcks HM, Rosenberger JG, Cox BM (
1991
) Nicotine indirectly inhibits [3H]-dopamine uptake at concentrations that do not directly promote [3H]-dopamine release in rat striatum.
J Neurochem
 
56
:
603
–610.
Lena C, Cangeux J P (
1997
) Role of Ca2+ ions in nicotinic facilitation of GABA release in mouse thalamus.
J Neurosci
 
17
:
576
–585.
Letz B, Schomerus C, Maronde E, Korf HW, Korbmacher C (
1997
) Stimulation of nicotinic Ach receptor causes depolarization and activation of L-type Ca2+ channels in rat pinealocytes.
J Physiol Lond
 
499
:
329
–340.
Lobron C, Wevers A, Damgen K, Jeske A, Rontal D, Birtsch C, Heinemann S, Reinhardt S, Schroder H (
1995
) Cellular distribution in the rat telencephalon of mRNA encoding for the alpha 3 and alpha 4 subunits of the nicotinic acetylcholine receptor.
Brain Res Mol Brain Res
 
30
:
70
–76.
Luetje CW, Patrick J (
1991
) Both alpha-and beta-subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors.
J Neurosci
 
11
:
837
–845.
Margiotta JF, Gurantz D (
1989
) Changes in the number, function and regulation of nicotinic acetylcholine receptors during neuronal development.
Dev Biol
 
135
:
326
–339.
Marquez FJ, Quesada AR, Sanchez-Jimenez F, Nuñez de Castro I (
1986
) Determination of 27 dansyl amino acid derivates in biological fluids by reversed-phase high-perfomance liquid chromatography.
J Chromatogr
 
380
:
275
–283.
Marshall D, Soliakov L, Redferm P, Wonnacott S (
1996
) Tetrodotoxin-sensitivity of nicotine-evoked dopamine release from rat striatum.
Neuropharmacology
 
35
:
1531
–1536.
Marshall D, Redfern P, Wonnacott S (
1997
) Presynaptic nicotinic modulation of dopamine release in the three ascending pathways studied by in vivo microdyalisis: comparison of native and chronic nicotine-treated rats.
J Neurochem
 
68
:
1511
–1519.
McGehee DS and Rolen LW (
1995
) Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons.
Annu Rev Physiol
 
57
:
521
–546.
McGehee DS, Health MJS, Gelber S, Devay P, Role LW (
1995
) Nicotine enhancement of rats excitatory synaptic transmission in CNS by presynaptic receptors.
Science
 
269
:
1692
–1696.
Miller C, Myles-Worsley M, Nagamoto HT, Rollins Y, Steven KE, Waldo M, Freedman R (
1996
) Nicotinic receptor function in schizophrenia.
Schizphrenia Bull
 
22
:
431
–445.
Mulle C, Choquet D, Korn H, Changeux J P (
1992
) Calcium influx through nicotinic receptor in rat central neurons: its relevance to cellular regulation.
Neuron
 
8
:
135
–143.
Nakayama H, Shioda S, Okuda K, Nakashima T, Nakai Y. (
1995
) Inmunocytochemical localization of nicotinic acetylcholine receptor in rat cerebral cortex.
Brain Res Mol Brain Res
 
32
:
321
–328.
Papke RL, Boulter J, Patrick J, Heinemann S (
1989
) Single-channel currents of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.
Neuron
 
3
:
589
–596.
Rust G, Burgunder JM, Lauterburg TE, Cachlin AB (
1994
) Expression of neuronal nicotinic acetylcholine receptor subunit genes in the rat autonomic nervous system.
Eur J Neurosci
 
6
:
478
–485.
Sargent PB (
1993
) The diversity of neuronal nicotinic acetylcholine receptors.
Annu Rev Neurosci
 
16
:
403
–483.
Schroder H, De Vos RA, Jansen EN, Birtsch C, Wevers A, Lobron C, Schroder R, Maelicke A (
1995
) Gene expression of the nicotinic acetylcholine receptor alpha 4 subunit in the frontal cortex in Parkinson's disease patients.
Neurosci Lett
 
187
:
73
–176.
Segal M (
1983
) Rat hippocampal neurons in culture: responses to electrical and chemical stimuli.
J Neurophysiol
 
50
:
1249
–1264.
Seguela P, Wadiche J, Dineley-Miller K, Dani JA, Patrck JW (
1993
) Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium.
Neuroscience
 
13
:
596
–604.
Slotkin TA, McCook EC, Seidler FJ (
1997
) Cryptic brain cell injury caused by fetal nicotine exposure is associated with persistent elevations of c-fos protooncogene expression.
Brain Res
 
750
:
180
–188.
Thoth E, Serhen H, Hashim A, Vizi E, Lajtha A (
1992
) Effect of nicotine on extracellular levels of neurotransmitters assessed by microdialysis in various brain regions: role of glutamic acid.
Neurochem Res
 
17
:
265
–271.
Thoth E, Vizi ES, Lajtha A (
1993
) Effect of nicotine on levels of extracellular amino acids in regions of the rat brain in vitro.
Neuropharmacology
 
32
:
827
–832.
Vernino S, Amador M, Luetje CW, Patrick J, Dani JA (
1992
) Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors.
Neuron
 
8
:
127
–134.
Vernino S, Rogers M, Radcliffe K, Dani JA (
1994
) Quantitative measurement of calcium flux through muscle and neuronal nicotinic acetylcholine receptors.
J Neurosci
 
14
:
5514
–5524.
Vidal C, Cangeux JP (
1989
) Pharmacological profile of nicotinic acetyl-choline receptors in the rat prefrontal cortex: an electrophysiological study in a slice preparation.
Neuroscience
 
29
:
261
–270.
Wada E, Wada K, Boulter J, Deneris E, Heinemann S et al. (
1989
) Distribution of α2, α3, α4 and β2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: hybridization histochemical study in the rat.
J Comp Neurol
 
284
:
314
–335.
Wada E, McKinnon D, Heinemann S, Patrick J, Swanson LW (
1990
) The distribution of mRNA encoded by a new member of the neuronal nicotinic acetylcholine receptor gene family (α5) in the rat central nervous system.
Brain Res
 
526
:
46
–53.
Waggoner AS (
1979
) Dye indicators of membrane potential.
Rev Biophys Bioenerg
 
8
:
47
–68.
Waniewski RA, Martin DL (
1994
) Acetylcholine receptor agonists stimulate [3H]-taurine release from rat sympathetic ganglia.
Eur J Pharmacol
 
260
:
113
–120.
Wilkie GI, Hutson P, Sullivan JP, Wonnacott S (
1996
) Pharmacological characterization of a nicotinic autoreceptor in rat hippocampal synaptosomes.
Neurochem Res
 
21
:
1141
–1148.
Zamanim MR, Allen YS, Owen GP (
1997
) Nicotine modulates the neurotoxic effect of beta-amyloid protein (25-35) in hippocampal cultures.
NeuroReport
 
8
:
513
–517.