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Keywords:

  • catecholamine outflow;
  • brain slice;
  • hippocampus;
  • neocortex;
  • corpus striatum;
  • electrical field;
  • nicotinic ACh receptors;
  • null mutation;
  • α5 subunit;
  • β2 subunit

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

Background and purpose:

Mice with targeted deletions of neuronal nicotinic acetylcholine receptor (nAChR) subunit genes are valuable models to study nAChR function such as catecholamine outflow by presynaptic receptor activation. Contrary to the rat, our present knowledge on presynaptic nAChRs in mice primarily relies on observations made with synaptosomes. We have now used brain slices to investigate nicotine-induced catecholamine outflow in wild type (WT) and nAChR (β2 and α5) knockout mice for a comparison with rat brain slice preparations.

Experimental approach:

Brain slices from rat and mouse hippocampus, parieto-occipital neocortex, and corpus striatum were loaded with either [3H]-noradrenaline or [3H]-dopamine. We provoked catecholamine outflow by electrical field stimulation and nicotinic agonists.

Key results:

When set in relation to electrical field stimulation, nicotine-evoked catecholamine release was sizeable in the striatum but low in the neocortex of both rats and mice. [3H]-noradrenaline outflow was, on the other hand, substantial in the rat but low in the mouse hippocampus. About 10% (or less) of nicotine-induced catecholamine release persisted in the presence of tetrodotoxin in all our preparations.

Conclusions and implications:

Targeted deletion of the β2 subunit gene essentially abolished the effect of nicotine, indicating that this subunit is an essential constituent of nAChRs that indirectly (via action potentials) induce catecholamine release from hippocampal and striatal slices in mice. The impact of nAChRs in catecholaminergic projection areas differs between species and has thus to be considered when extrapolating results from animal models to human conditions.

British Journal of Pharmacology (2007) 151, 414–422; doi:10.1038/sj.bjp.0707236


Abbreviations:
α-CtxMII

α-Contotoxin MII

DMPP

1,1,-dimethyl-4-phenylpiperazinium

KO

knockout

nAChR

nicotinic acetylcholine receptor

Rmax

maximally induced release

TTX

tetrodotoxin

WT

wild type

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

Nicotinic acetylcholine receptors (nAChRs) modulating the release of neurotransmitters from presynaptic terminals have a definite role in the central nervous system (reviewed by Role and Berg, 1996; Wonnacott, 1997). Our knowledge of these receptors rests to a large extent on the neurochemical analysis of transmitter release from either synaptosomes or brain slices. In contrast to synaptosomes, brain slices retain local anatomical integrity which may account for significantly differing results between the two preparations (Kaiser et al., 1998; Wonnacott et al., 2000; Leslie et al., 2002). [3H]dopamine release is, for instance, evoked by anatoxin-a with two affinities (0.24 and 5.1 μM) in rat striatal slices, whereas a single EC50 (0.13 μM) was determined in striatal synaptosomes (Kaiser and Wonnacott, 2000; Wonnacott et al., 2000). Likewise, the sensitivity to tetrodotoxin (TTX) of nicotine-induced catecholamine release differs significantly between the two preparations (reviewed in Wonnacott, 1997). To appreciate functions of nAChRs in terminal fields it is thus essential to supplement results on nAChR activation in synaptosomes with observations from brain slice preparations.

Although previous work allows such comparison for the rat, neurochemical studies of presynaptic nAChRs in mice have primarily taken advantage of synaptosomal preparations (Marks et al., 1993, 1995, 2000; Grady et al., 2002; Champtiaux et al., 2003; Salminen et al., 2004; Azam and McIntosh, 2006). We have now set out to analyse catecholamine outflow from hippocampal, neocortical and striatal brain slices of wild-type (WT) C57BL/6J mice. The impact of the β2 subunit on nicotine-evoked catecholamine outflow was investigated in the hippocampus and the striatum taken from animals with a targeted deletion of the nAChR β2-subunit gene (Picciotto et al., 1995). We used, in addition, α5-knockout (KO) mice (Salas et al., 2003) to explore the function of this subunit in striatal slice preparations. Our results confirm previous observations on the crucial role the β2 subunit plays in nicotine-induced catecholamine release from the hippocampus as well as the striatum. However, and unlike the peripheral nervous system where a null mutation of the α5-subunit gene dramatically enhances nicotine-evoked [3H]noradrenaline outflow (Fischer et al., 2005), this KO had no significant effect on [3H]dopamine release from striatal slices.

The catecholamine outflow evoked by nAChR activation in mouse brain slice preparations is mainly caused by an indirect (TTX-sensitive) component. In hippocampal and striatal slices, this component critically depends on the presence of the nAChR β2 subunit. Our comparative studies with rat brain slice preparations indicate major species and regional differences of catecholamine release in response to nAChR activation.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

Animals

About 3–4-week-old male Sprague–Dawley rats were obtained from the Institute of Biomedical Research, Medical University of Vienna (Himberg, Austria). WT-C57BL/6J mice and mice deficient in the α5 nAChR subunit gene were from Avi Orr–Urtreger (Salas et al., 2003). The β2-KO strain was from J-P Changeux (Picciotto et al., 1995). The two KO strains have been backcrossed onto a C57BL/6J background for seven and 12 generations, respectively. We used 6–8-week-old male mice bred from homozygous parent animals at our in-house animal facilities. All animals were kept in thermostable rooms (21°C) at a light–dark schedule of 22:14 h in-group cages, with food and water freely accessible.

Superfusion experiments

All animals were killed by CO2 anaesthesia and decapitation according to the Guidelines of the Animal Care Committee of the Medical University of Vienna. The brains were rapidly removed and placed in ice-cold superfusion buffer containing (mM): NaCl 118; KCl 4.8; CaCl2 2.5; MgSO4 1.2; NaHCO3 25; KH2PO4 1.2; Na2-ethylenediaminetetraacetic acid 0.03; glucose 11; ascorbic acid 0.57; fumaric acid 0.5; sodium pyruvate 5.0; saturated with 95% O2/5% CO2. Hippocampus, striatum and (parieto–occipital) neocortex were cut into 300 μm slices by means of a McIllwain tissue chopper. Loading of radioactivity was achieved by adding either 0.03 μM [3H]noradrenaline or 0.2 μM [3H]dopamine and 1 mM ascorbic acid to buffer, followed by incubations for 60 min at 36.5°C. The brain slices were then placed in small chambers between two platinum wire electrodes and superfused for 1 h with buffer (kept at 26°C and continuously bubbled with 95% O2/5% CO2) in the presence of 0.5 μM clorgyline. We collected 4 min fractions at a flow rate of 1 ml min−1.

Stimulated [3H] outflow was achieved by either adding nAChR agonists to the superfusion buffer for 30 s or by electrical field stimulation (100 pulses, 10 Hz, 0.5 ms, 50 V cm−1, 40 mA). Each slice was tested with a single application of a nicotinic agonist (see Figure 1a and d). Mecamylamine and TTX were added to the superfusion buffer 12 min before and during the stimulation with nicotine. By taking advantage of the slow off-kinetics, the effects of α-conotoxin MII (α-CtxMII) were tested by pre-exposing cultures for 12 min before applying a nicotine stimulus (Salminen et al., 2004). The radioactivity retained by slices at the end of an experiment was recovered by extraction with 1% sodium dodecyl sulphate and sonication.

image

Figure 1. [3H]noradrenaline outflow from mouse (a–c) and rat (d–f) hippocampal slices. (a) Fractional rates of outflow in response to electrical field stimulation (circles) or to the application of 100 μM nicotine (triangles). Data are means±s.e.m. (n=3). (b) [3H]noradrenaline outflow in response to indicated stimuli (shown as a percentage of total radioactivity in slices) in WT mice (n=6–27). (c) [3H]noradrenaline outflow in response to indicated stimuli in β2-KO mice (n=6–9). Compared to WT mice (b), the outflow induced by 20 μM nicotine was reduced by 95.2% in the KO. (d) Fractional rates of [3H]noradrenaline outflow in response to electrical field stimulation (circles) or to the application of 100 μM nicotine (triangles; n=3). (e) [3H]noradrenaline outflow in response to indicated concentrations of nicotine (calculated EC50: 19.6 μM; n=3). (f) [3H]noradrenaline outflow in response to indicated stimuli (n=3–12). nic, nicotine; TTX, 0.5 μM TTX; MCA, 5 μM mecamylamine.

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Calculations

Fractional rates of [3H] outflow were calculated by dividing the radioactivity of a 4 min fraction by the total radioactivity of slices at the beginning of the corresponding 4 min collection period. The total radioactivity at a given time was obtained by adding up the residual radioactivity retained by slices with the radioactivity of all fractions collected from this point. Stimulation-evoked outflow was calculated as the difference between the total [3H] outflow during and after stimulation on one hand, and the estimated basal outflow on the other hand, assuming that basal release follows a linear decline. Both fractional rates and stimulated outflow were multiplied by 100 and thus expressed as % of the total radioactive contents of a slice. Radioactivity in extracts and collected fractions was determined by liquid scintillation counting.

Data analysis

Data are shown as means±s.e.m. The significance of differences was evaluated with the unpaired Student's t-test, unless indicated otherwise. Concentration–response curves for agonists were fitted by unweighted non-linear regression to the logistic equation with GraphPad Prism software. The programme uses F statistics as the ‘extra sum of squares’ introduced when testing for independent (i.e. significantly different at P<0.05) versus shared (i.e. not significantly different) parameters of curves (EC50, slope, maximal release Rmax).

Materials

Materials and reagents were from the following sources: (−)-[ring-2,5,6 3H]noradrenaline (60–75 Ci mmol−1, PerkinElmer, Waltham, MA, USA); [3H]dopamine (9.9 Ci mmol−1, Amersham-Biosciences, GE-Healthcare, Chalfont St Giles, GB); α-CtxMII was synthesized as previously described (Cartier et al., 1996); TTX HCl (TTX: Latoxan, 26000 Valence – France). 1,1,-Dimethyl-4-phenylpiperazinium iodide (DMPP, D5891), (−)-nicotine (N3876), (−)-cytisine (C2899), mecamylamine hydrochloride (M9020), and clorgyline hydrochloride (M3778) were from Sigma-Aldrich (St Louis, MO, USA). Other chemicals were from Merck (Darmstadt, Germany), analytical grade.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

Hippocampal brain slice preparations from mice and rats

Electrical field stimulation caused a large outflow of [3H]noradrenaline from mouse hippocampal brain slices (Figure 1a–c) that was entirely abolished in the presence of 0.5 μM TTX (Figure 1b).

The nicotine-induced release was small by comparison (Figure 1a and b). Calculating a ratio of [3H]noradrenaline outflow in response to a maximally effective nicotine concentration (Figure 1b) and electrical field stimulation (Figure 1b) yielded a ‘standardized nicotine index’ of 0.13 (Table 1). The nicotine-induced [3H]noradrenaline outflow was largely prevented not only by adding the nicotinic antagonist mecamylamine (5 μM, Figure 1b) but also by the sodium channel blocker (TTX, 0.5 μM). In the presence of TTX, the [3H]noradrenaline outflow evoked by 20 μM nicotine amounted to just 0.034±0.022% (n=8) of the radioactive contents of the slices (Figure 1b).

Table 1. Standardized nicotine indices
 C57Bl/6J WT mouseRat
  1. Abbreviation: Wt, wild type.

  2. Standardized nicotine indices were obtained by dividing nicotine-induced catecholamine outflow (first number in parenthesis) by the outflow in response to electrical field stimulation of brain slices (second number in parenthesis). Data for this table were taken from experiments shown in Figures 1, 2 and 3 with a nicotine concentration yielding maximal outflow.

Hippocampus0.13 (1.25, 9.37%)0.81 (4.63, 5.70%)
Neocortex0.08 (0.77, 9.42%)0.21 (1.11, 5.17%)
Striatum0.93 (1.73, 1.86%)1.45 (0.90, 0.62%)

Targeted deletion of the β2 nAChR subunit gene essentially abolished all nicotine-evoked [3H]noradrenaline release in these mice, whereas the [3H]noradrenaline outflow in response to electrical field stimulation was unaffected (Figure 1c).

The impact of nAChR activation was quite different in rat hippocampal brain slices (Figure 1d–f) with a ‘standardized nicotine index’ of 0.81 (Table 1). Hence, 100 μM nicotine caused almost as much [3H]noradrenaline outflow as did electrical field stimulation (Figure 1f). We found nicotine EC50 values (19.6 μM; log EC50: −4.707±0.126) and maximal effects (6.9±0.92%; Figure 1e) in good agreement with a previous study (Leslie et al., 2002). As in mouse hippocampal brain slices, the nicotine-induced outflow was largely prevented in the presence of 0.5 μM TTX or 5 μM mecamylamine (Figure 1f).

As we used rats of younger age (3–4 weeks) than mice (6–8 weeks), and as age-dependent effects of nicotine on the outflow of [3H]noradrenaline from both hippocampal synaptosomes and slice preparations have been reported in either species (Leslie et al., 2002; Azam and McIntosh, 2006) we checked whether mice of similar age showed larger effects in response to nicotine. However, the outflow of [3H]noradrenaline induced by 100 μM nicotine did not differ significantly between 20, 48 and 70-days-old mice (20 days: 0.95±0.12%, n=9; 48 days: 1.17±0.13%; n=6; 70 days: 0.95±0.19%, n=9; P>0.05, one-way analysis of variance).

Neocortical brain slice preparations from mice and rats

The [3H]noradrenaline outflow from mouse (parieto–occipital) neocortical slices was similar to the mouse hippocampus: high in response to electrical field stimulation, but low upon nAChR activation (Figure 2a), yielding a ‘standardized nicotine index’ of just 0.08 (Table 1).

image

Figure 2. [3H]noradrenaline outflow from mouse (a) and rat (b, c) parieto–occipital neocortical slices. (a) [3H]noradrenaline outflow in response to indicated stimuli (shown as a percentage of total radioactivity). Data are means±s.e.m. (n=3–6). (b) [3H]noradrenaline outflow in response to indicated concentrations of nicotine (EC50: 7.8 μM; n=3). (c) [3H]noradrenaline outflow in response to indicated stimuli (n=5–15). nic, nicotine; TTX, nicotine in the presence of 0.5 μM TTX; MCA, nicotine in the presence of 5 μM mecamylamine.

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In rat neocortical slices, nicotine was about as potent (EC50, 7.8 μM; log EC50, −5.107±0.585) as in hippocampal slices, though clearly less efficient in inducing [3H]noradrenaline outflow (Figure 2b). Owing to a sizeable outflow in response to electrical field stimulation, the small effect of a maximally effective nicotine concentration resulted in a ‘standardized nicotine index’ of 0.21 (Figure 2c; Table 1). TTX and mecamylamine effectively inhibited nicotine-induced [3H]noradrenaline outflow in both mice (Figure 2a) and rats (Figure 2c).

Striatal brain slice preparations from mice and rats

[3H]dopamine outflow from mouse striatal slice preparations in response to electrical field stimulation (Figure 3b) was significantly smaller than [3H]noradrenaline outflow from either the hippocampus (Figure 1b) or the neocortex (Figure 2a). Likewise, electrical field stimulation of rat brain slices caused a significantly smaller outflow of [3H]dopamine from the striatum (Figure 3e) than [3H]noradrenaline outflow from the hippocampus (Figure 1f) or the neocortex (Figure 2c).

image

Figure 3. [3H]dopamine outflow from mouse (a–c) and rat (d–e) corpus striatum slices. (a) [3H]dopamine outflow (shown as a percentage of total radioactivity) in response to 10 μM nicotine and indicated experimental conditions. Each approach was run with its own set of [3H]dopamine outflow evoked by 10 μM nicotine at control conditions. Data are means±s.e.m. (n=3–15). (b) [3H]dopamine outflow in response to electrical field stimulation (n=16) or 10 μM nicotine (n=17) in WT mice. (c) [3H]dopamine outflow in response to electrical field stimulation (n=11) or 10 μM nicotine (n=6) in β2-KO mice. (d) [3H]dopamine outflow in response to indicated concentrations of nicotine (EC50, 0.84 μM; n=3–6). (e) [3H]dopamine outflow in response to indicated stimuli (n=3–9). nic, nicotine; TTX, 0.5 μM TTX; no Ca2+, Ca2+ omitted before and during nicotine application; MCA, 1 and 3 μM mecamylamine; MII, 50 and 100 nMα-CtxMII.

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The lower [3H]dopamine outflow in response to electrical field stimulation was not paralleled by a reduced effect of nicotine in the striatum, resulting in a ‘standardized nicotine index’ of 0.93 (mouse) and 1.45 (rat) (Figure 3b and e; Table 1). In mice, 50 μM nicotine-evoked [3H]dopamine outflow from striatal slices (Figure 3b) was comparable to the nicotine-induced [3H]noradrenaline release from the hippocampus (Figure 1b) and significantly larger than in the neocortex (Figure 2a). In line with previous reports (Sacaan et al., 1995; Wonnacott et al., 2000), nicotine-induced [3H]noradrenaline outflow from rat striatal slices with high potency (EC50, 0.84 μM; log EC50, −6.073±0.332) but low efficacy (maximal effect: 1.3±0.8%; Figure 3d).

The rather large ‘standardized nicotine index’ in mice suggested the possibility that [3H]dopamine outflow in response to electrical field stimulation was caused, in part, by ACh released from cholinergic neurons. We tested this hypothesis by adding 5 μM mecamylamine between two electrical stimuli set 20 min apart to our superfusion buffer. In the absence of MCA, the second stimulus was 80.6±12.8% (means±s.e.m., n=10) of the first stimulus. In the presence of 5 μM mecamylamine (added 12 min before and during stimulation), the second stimulus was 80.4±9.4% (n=12). Hence, activation of nAChRs by endogenous ACh did not significantly contribute to [3H]dopamine outflow in our paradigm.

The nicotine-induced [3H]dopamine outflow from mouse striatal slices was examined in more detail. It was entirely dependent on the presence of calcium in the superfusion buffer (Figure 3a), largely reduced in the presence of 0.5 μM TTX (Figure 3a), blocked by mecamylamine in a concentration-dependent manner (Figure 3a), and partially inhibited by α-CtxMII (to 50.2 and 54.8% in the presence of 50 and 100 nMα-CtxMII, respectively, Figure 3a). Null mutation of the β2 nAChR subunit gene almost abolished [3H]dopamine outflow in response to nicotine (Figure 3c). Electrically induced outflow was, on the other hand, not significantly affected by the KO (Figure 3b and c).

Null mutation of the α5 nAChR subunit gene has been reported to affect ACh-induced [3H]dopamine outflow from mouse striatal synaptosomes (Salminen et al., 2004). We thus compared the [3H]dopamine outflow in response to nicotine, cytisine and 1,1,-dimethyl-4-phenylpiperazinium iodide (DMPP) from striatal slices of WT and α5-KO animals. However, our experiments revealed no significant difference of either potencies or efficacies between the two genotypes (Figure 4; Table 2).

image

Figure 4. [3H]dopamine outflow in response to nicotine (a), DMPP (b) and cytisine (c) from striatal slices taken from WT (circles, solid lines) or α5-KO mice (triangles, dashed lines). Data points are means±s.e.m. (n=3–15). Curves were fitted to data points as described in Methods. See Table 2 for EC50 and Rmax values.

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Table 2. Agonist-induced [3H]DA outflow from striatal slices of WT and α5-KO mice
 EC50 (log EC50)Rmax (S %)
  1. Abbreviations: DMPP, 1,1,-dimethyl-4-phenylpiperazinium; KO, knockout; WT, wild type.

  2. EC50 (log EC50±s.e.m.) and maximal release Rmax are (±s.e.m.) obtained by fitting concentration–response curves to data points shown in Figure 4. Fit parameters (EC50 and Rmax) did not differ significantly between WT and KO animals for any of the three agonists (P>0.05, see Methods for statistics applied).

Nicotine, WT0.80 μM (−6.09±0.270)2.85±0.41
Nicotine, KO1.00 μM (−5.99±0.197)2.26±0.27
DMPP, WT2.57 μM (−5.592±0.175)2.58±0.40
DMPP, KO7.59 μM (−5.120±0.360)2.78±0.56
Cytisine, WT0.46 μM (−6.336±0.652)1.15±0.45
Cytisine, KO0.14 μM (−6.827±0.230)0.86±0.12

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

The object of this study was to characterize properties of nAChRs triggering catecholamine release in brain slice preparations of mice. We furthermore compared the two species, rats and mice, and the outflow following two types of stimulus, nicotine and electrical field stimulation. The principal findings of our study confirm and significantly extend previous observations made on mouse synaptosomes and indicate major species and regional differences of catecholamine outflow: (1) nicotine-induced [3H]noradrenaline outflow from hippocampal slices was significantly larger in rats than in mice. (2) Irrespective of species, electrical field stimulation of slices produced significantly more [3H]noradrenaline release from both the hippocampus and the neocortex than [3H]dopamine outflow from the striatum. (3) The nicotine-evoked catecholamine release was largely prevented in all slice preparations by the presence of TTX. (4) Nicotine-induced [3H]dopamine outflow from the striatum and [3H]noradrenaline release from the hippocampus of mice required the presence of the β2nAChR subunit. (5) Targeted deletion of the α5 subunit gene had no significant effect on [3H]dopamine outflow from the mouse striatum in response to nAChR activation.

The modulation [3H]noradrenaline outflow from hippocampal slices by nAChR activation was species dependent

The robust outflow of [3H]noradrenaline in response to nAChR activation in rat hippocampal slices is well documented (Sacaan et al., 1995, 1996; Sershen et al., 1997; Lena et al., 1999; Leslie et al., 2002; Amtage et al., 2004). By comparison with our standard electrical field protocol, we found about equal quantities of [3H]noradrenaline release in response to stimulation with maximally effective nicotine concentrations (‘standardized nicotine index’: 0.81, Table 1).

In contrast, this ratio diminished to 0.13 in mouse hippocampal slices, indicating that nAChR activation triggers NA release less efficiently in the mouse than in the rat hippocampus. This observation is in line with a recent report showing that nicotine-evoked [3H]noradrenaline outflow from hippocampal synaptosomes is larger in rats than in mice (Azam and McIntosh, 2006). Such species difference has also been reported for the neocortex, where [3H]noradrenaline outflow induced by nicotine was significantly larger in human than in rat brain slice preparations (Amtage et al., 2004). Whether properties of these nAChRs in the human hippocampus compare better with the mouse than with the rat is not known. Species differences in nAChRs become critical though when applying observations from animal models to neuropathological mechanisms in men (Kaiser and Wonnacott, 2000; Picciotto et al., 2001; Amtage et al., 2004).

The impact of nAChR activation on catecholamine outflow differs between brain regions

Transmitter outflow in response to electrical field stimulation is the sum of several parameters, such as the number of presynaptic specializations, reuptake and degradation mechanisms and feedback inhibition by presynaptic autoreceptors (Valenta et al., 1988; Boehm and Huck, 1995). Any of these parameters might contribute to the larger [3H]noradrenaline release from neocortex and hippocampus compared to a smaller [3H]dopamine outflow from the striatum. The smaller effect of electrical field stimulation on [3H]dopamine outflow in the striatum did not go together with a reduction of nicotine effects. Hence, indices for nicotine-induced [3H]dopamine outflow set in relation to electrical field stimulation in mice yielded 0.93 (striatum), opposed to 0.13 (hippocampus, Table 1). These data, and the indices calculated for rat neocortical, hippocampal and striatal slices (0.21, 0.81 and 1.45, respectively, Table 1) suggest that the impact of nAChR activation on catecholamine release distinctly differs in these structures.

Indirect mechanisms of nAChR-modulated catecholamine release markedly outweigh direct mechanisms

Our finding that TTX inhibited nicotine-evoked catecholamine outflow in all our preparations by about 90% are in line with previous observations made on human and rat brain slice preparations (Sacaan et al., 1995; Marshall et al., 1996; Wonnacott, 1997; Lena et al., 1999; Leslie et al., 2002; Amtage et al., 2004; Barik and Wonnacott, 2006). Quite the opposite, nicotinic agonist-induced catecholamine release from rat synaptosomes is clearly less sensitive to an inhibition by TTX (Soliakov et al., 1995; Clarke and Reuben, 1996; Marshall et al., 1996; Leslie et al., 2002). To our knowledge, effects of TTX on nicotine-induced catecholamine release have not been studied in mouse synaptosomal preparations, though [86Rb+] efflux from mouse thalamic synaptosomes in response to nicotine was partly inhibited (by 42%) in the presence of TTX (Marks et al., 1995).

A sizeable effect of TTX indicates that a major part of nAChRs, though still on the catecholaminergic axon, is too far distant from active zones to trigger exocytosis directly by calcium influx through the nAChR channel pore (Wonnacott, 1997; Kristufek et al., 1999). As even synaptosomal preparations may show some TTX sensitivity (Soliakov et al., 1995; Marshall et al., 1996), nAChRs that trigger catecholamine release by a direct (TTX-resistant) and an indirect (TTX-sensitive) mechanism may coexist in relative close proximity (i.e., on isolated synaptosomes).

nAChRs modulating catecholamine outflow indirectly may in addition reside on glutamatergic axons, as shown in rat striatal slice preparations (Wonnacott et al., 2000; Kaiser and Wonnacott, 2000). Indirect effects of nAChR activation are more complex in the hippocampus, where both GABA-ergic and glutamatergic mechanisms are involved (Leslie et al., 2002; Barik and Wonnacott, 2006). In the rat, these indirect effects appear to be mediated by α7 nAChRs (Kaiser and Wonnacott, 2000; Wonnacott et al., 2000; Barik and Wonnacott, 2006).

Our experiments revealed that nicotine-evoked [3H]dopamine outflow from mouse striatal slices was reduced to 6% (compared to controls) in animals lacking the βnAChR subunit. These observations are in line with data obtained in striatal synaptosomal preparations, where β2 null mutations were found to eliminate nicotine-induced [3H]dopamine release (Whiteaker et al., 2000; Grady et al., 2002; Champtiaux et al., 2003; Salminen et al., 2004). However, our results imply that not only direct (TTX-resistant) but also indirect mechanisms depend on nAChRs containing the β2 subunit, as most of the [3H]dopamine outflow from mouse striatal slices in response to nicotine was owing to an indirect (TTX-sensitive) effect. nAChRs containing the β2 subunit also mediate the indirect (action potential-dependent) dopamine release caused by endogenous ACh in striatal slice preparations (Zhou et al., 2001).

We have shown that β2 null mutations greatly reduced catecholamine outflow not only from striatal but also from hippocampal slices. These data are in line with a recent study showing that [3H]noradrenaline outflow in response to nicotine is abolished in hippocampal synaptosomes prepared from β2-KO animals (Azam and McIntosh, 2006). By the combined use of subunit-specific conotoxins (α-MII, α-BuIA, α-PIA and α-AuIB) and KO animals (β2, β3, β4 and α4) the authors identified two types of nAChRs modulating [3H]noradrenaline outflow from synaptosomes: α6(α4)β2β3β4 and α6(α4)β2β3.

Pharmacological evidence suggests that receptors modulating [3H]noradrenaline outflow in rat hippocampal synaptosomes are distinct from those in mice and contain the subunits α3 and β4 (Clarke and Reuben, 1996; Luo et al., 1998; Azam and McIntosh, 2006). Hence, α-Ctx MII fully inhibits [3H]noradrenaline outflow in mice but is ineffective in rat synaptosomes. Likewise, α-Ctx PIA which reduces [3H]noradrenaline outflow by about 80% in mice potentiates nicotine effects in rat hippocampal synaptosomes (Azam and McIntosh, 2006)). The significantly larger outflow of [3H]noradrenaline in response to nicotine seen in rat compared to mouse hippocampal synaptosomes (Azam and McIntosh, 2006) may thus be caused by more efficient receptors, a higher number of receptors, or both.

Synaptosomes taken from adult mice do not display significant [3H]noradrenaline outflow above baseline (Azam and McIntosh, 2006). As most of our experiments were performed on hippocampal slices of 6–8 weeks old mice, nAChRs modulating [3H]noradrenaline release by indirect mechanisms not residing on noradrenergic projections may play a dominant role. The absence of nicotine-evoked [3H]noradrenaline outflow from hippocampal slices taken from β2-KO animals indicates that in mice, these indirect mechanisms crucially depend on the presence of the β2 subunit.

Targeted deletion of the nAChR α5 subunit gene

Three main types of hetero-oligomeric nAChRs have been identified in mouse dopamine terminal fields: α4β2*, α6β2* and α4α6β2*. Whereas α6-containing receptors are sensitive to α-CtxMII, those made of α4β2* are not (Champtiaux et al., 2003). α5 was present in 9% of purified α4* nAChRs but in only 1% of α6* receptors (Champtiaux et al., 2003). Nonetheless, targeted deletion of the α5 subunit gene affected not only α-CtxMII-insensitive (presumably mediated by α4β2* receptors) but also α-CtxMII-sensitive (presumably mediated by α6* receptors) ACh-induced outflow of [3H]DA, albeit in an opposite manner: Whereas maximally induced release (Rmax) in the presence of 100 nMα-CtxMII was reduced in the α5 KO, the Rmax of the α-CtxMII-sensitive component was enhanced (Salminen et al., 2004). The report makes no mention whether the two opposite effects of the α5-KO level off when looking at overall outflow in the absence of α-CtxMII.

As in synaptosomal preparations (Kulak et al., 1997; Kaiser et al., 1998; Kaiser and Wonnacott, 2000; Grady et al., 2002; Champtiaux et al., 2003; Salminen et al., 2004) we found α-CtxMII to inhibit [3H]dopamine release from mouse striatal slices by about 50% in response to nicotine. Our assay is, however, not sufficiently robust to distinguish possible effects of a deletion of the α5 subunit on the α-CtxMII-sensitive and the α-CtxMII-insensitive component of [3H]dopamine release, as carried out in mouse striatal synaptosomes (Salminen et al., 2004). The overall release (Rmax and EC50 values in the absence of α-CtxMII) induced by three nicotinic agonists nicotine, cytisine and DMPP was not significantly affected in mice lacking the α5 subunit. These results are in contrast to observations we made in cultured sympathetic neurons, where targeted deletion of the α5 subunit gene caused a large enhancement of nicotine-evoked [3H]noradrenaline outflow (Fischer et al., 2005).

Impact of nAChRs located in the projection areas of catecholaminergic neurons

Excitation of dopaminergic neurons in the midbrain and of noradrenergic neurons in the locus coeruleus play a major, if not dominant, role in the release of catecholamines upon systemic application of nicotine (Mitchell, 1993; Fu et al., 1998; Mansvelder and McGehee, 2000; Picciotto and Corrigall, 2002; Mansvelder et al., 2003; David et al., 2006). The collective activation of projecting axons by electrical field stimulation in brain slices provides some indirect evidence on the overall release capacity of these systems. When set in relation to the electrically induced outflow, nicotine by acting on receptors in the terminal fields is about equi-effective in stimulating [3H]dopamine release from the striatum, but significantly less so in evoking [3H]noradrenaline outflow from the neocortex of both rats and mice. The modulation of [3H]noradrenaline outflow in the hippocampus by nicotine is species-dependent: sizeable in rat, but small in the mouse hippocampal slice preparation.

Our comparative observations made in striatal, hippocampal and neocortical slice preparations from rats and mice showed regional and species dependent differences of nAChR properties in the terminal fields of catecholaminergic projections. In mice, nicotine-evoked catecholamine outflow is primarily mediated by action potentials and dependent on nAChRs containing the β2 subunit.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References

Expert technical assistance was provided by Gabriele Koth and Karin Schwarz. This work was supported by a Grant GZ 70.072/2-PR/4/2000 (Austrian Ministry for Education, Science and Culture to SH) and NIH MH53631 (to JMM).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of interest
  9. References
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