Characterization of nicotinic receptors involved in the release of noradrenaline from the hippocampus

Pergamon

PII:

Neuroscience Vol. 77, No. 1, pp. 121–130, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/97 $17.00+0.00 S0306-4522(96)00425-3

CHARACTERIZATION OF NICOTINIC RECEPTORS INVOLVED IN THE RELEASE OF NORADRENALINE FROM THE HIPPOCAMPUS H. SERSHEN,* A. BALLA,† A. LAJTHA* and E. S. VIZI†‡ *Center of Neurochemistry, The Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962, U.S.A. †Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O. Box 67, H-1450 Budapest, Hungary Abstract––The pharmacological features of putative nicotinic acetylcholine receptor sites involved in the release of [3H]noradrenaline were assessed in rat hippocampus. The effect of nicotinic agonists to induce [3H]noradrenaline release was examined in superfused slices. The nicotinic agonists (")-epibatidine, (+)-anatoxin-a, dimethylphenylpiperazinium, (")-nicotine and (")-lobeline released [3H]noradrenaline. The dose–response curves to nicotinic agonists were bell shaped, and indicated that their functional efficacies and potency vary across agonists. Maximal efficacy was seen with dimethylphenylpiperazinium and lobeline (Emax values two to three times higher than other agonists). The rank order of potency for the agonists to release [3H]noradrenaline was (")-epibatidine>(+)anatoxin-a>dimethylphenylpiperazinium>cytisine>nicotine>(")-lobeline. The nicotinic acetylcholine receptor antagonists (n-bungarotoxin>mecamylamine>(+)-tubocurarine>hexamethonium±á-bungarotoxin=dihydro-â-erythroidine) and tetrodotoxin antagonized the effect of dimethylphenylpiperazinium to release [3H]noradrenaline. The results, based on these pharmacological profiles, suggest the possible involvement of á3 and â2 nicotinic acetylcholine receptor subunits in the control of [3H]noradrenaline release from hippocampal slices. The absence of effect of á-bungarotoxin and á-conotoxin-IMI excludes the possible involvement of nicotinic acetylcholine receptors containing the á7 subunit. The release of [3H]noradrenaline by dimethylphenylpiperazinium was Ca2+ dependent. Nifedipine failed to prevent the dimethylphenylpiperazinium-induced release of [3H]noradrenaline, but Cd2+, ù-conotoxin and Ca2+-free conditions significantly reduced the dimethylphenylpiperazinium-induced release, suggesting that N-type voltage-sensitive Ca2+ channels are involved in the nicotinic acetylcholine receptor response. These voltage-sensitive Ca2+ channels are activated by the local depolarization produced by sodium influx through the nicotinic channels activated by dimethylphenylpiperazinium. Thus, the observed tetrodotoxin sensitivity of dimethylphenylpiperazinium-induced release of [3H]noradrenaline can be explained either by local depolarization and subsequent generation of action potentials at the preterminal area or that these nicotinic acetylcholine receptors are located on interneurons rather than directly on noradrenergic terminals. Copyright ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: noradrenaline release, nicotinic acetylcholine receptor, subtypes, noradrenergic receptor, presynaptic receptors, hippocampus.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated cation channels. Their activation facilitates the release of many neurotransmitters in the brain, including acetylcholine (ACh),3,32 noradrenaline (NA),11 serotonin12 and dopamine.9,29 Mesolimbic dopamine neurons can be influenced by release of ACh acting on nicotinic and muscarinic receptors, and nigrostriatal dopaminergic neurons can be influenced by ACh acting primarily on muscarinic receptors,10 but also on nAChRs.8,9,29,36 ‡To whom correspondence should be addressed. Abbreviations: ACh, acetylcholine; DG, dentate gyrus; DHâE, dihydro-â-erythroidine; DMPP, dimethylphenylpiperazinium; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; FRR, fractional resting release; NA, noradrenaline; nAChR, nicotinic acetylcholine receptor. 121

The hippocampus contains a high density of cholinergic innervation.16 We recently showed that noradrenergic and cholinergic axon terminals in the hippocampal regions are not equipped with inhibitory á2-autoreceptors and muscarinic heteroceptors, based on the finding that the á2adrenoceptor and the muscarinic receptor agonists/ antagonists did not affect the release of NA and ACh, respectively.22 The failure of atropine to increase and oxotremorine to reduce the release of NA indicated that cholinergic and adrenergic afferents do not communicate with each other presynaptically, at least not in an inhibitory manner.22 However, we still do not know whether the noradrenergic axon terminals in the hippocampus are equipped with stimulatory nicotinic receptors. If this is the case, it would provide a possibility for the septohippocampal

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cholinergic pathway, otherwise important in modulating memory formation, to functionally establish an excitatory connection with noradrenergic neurons in the hippocampus through nAChRs. Furthermore, nAChRs are multimeric proteins composed of homologous subunits, an agonistbinding á subunit and a structural â subunit. We examined whether the subclassification, based on the selectivity of agonists, antagonists and neurotoxins in radioligand binding and molecular biology experiments,19–21,27,49 holds true in experiments when the nAChRs were studied using neurochemical methods. The extensive diversity of the nAChR subtypes suggests additional diversity in their function to modulate neurotransmitter release. Such functional diversity has been characterized with selective nicotinic agonists, for example the á4â2 and á7 subtypes that mediate ACh release in the hippocampus. These subtypes show selectivity to the nicotinic agonist cytisine.5 The present study characterized the pharmacological features of putative nAChR sites involved in the dimethylphenylpiperazinium (DMPP)-induced release of NA from the rat hippocampus. In addition, an attempt was made to identify the calcium channel involved in the release process induced by nAChR stimulation. Some of these results have been communicated to the American Society for Neurochemistry.46

EXPERIMENTAL PROCEDURES

Preparation of subregions of hippocampal slice Anesthetized Sprague–Dawley adult rats (140–160 g; Charles River) were used. Hippocampal tissue was dissected out and sliced at 0.4 mm with a McIlwain tissue slicer. The slices were separated by shaking in ice-cold modified Krebs buffer. The whole-slice preparation was used, but in some initial experiments tissue slices were further dissected into CA1, CA3 and dentate gyrus (DG) regions under a lowpower microscope.22 Although there is a marked regional difference in distribution of noradrenergic innervation within the hippocampus,14,22 there were no significant differences in the effect of DMPP to release [3H]NA from the noradrenergic axon terminals in the CA1, CA3 and DG regions of the hippocampus. Therefore, whole hippocampal slice preparations were used for further identification of the subunit composition of nAChRs involved in the release of NA by the nAChR agonist DMPP. After dissection, the slices or subregions were incubated for 45 min in 1 ml of buffer containing [3H]NA (10 µCi/ml, 40 Ci/mmol; Amersham). Experiments were carried out at 37)C in a modified Krebs solution containing (mM): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25 and glucose 12.5, continuously saturated with carbogen gas (95% O2+5% CO2). After incubation, the slices or subregions (10 pieces of the CA1 and 15 pieces of the CA3 and DG regions; approximately 3 mg tissue weight) were transferred to a thermoregulated Plexiglas tissue chamber with an inside volume of 100 µl. The slices were perfused at a rate of 0.5 ml/min. The perfusate collected during the first 60 min was discarded, and subsequent superfusion fluid was collected in 1.5-ml (3-min) fractions for an additional 60 min. Antagonists/agonists were added at the eighth collection period. In those experiments in which the effect of

antagonists was studied, DMPP (20 mM) was added at the 10th collection period and maintained until the end of the experiment. At the end of the perfusion, the tissue was removed from the perfusion chamber and suspended in 500 µl of 10% trichloroacetic acid. An aliquot (100 µl) was assayed for tissue radioactivity. To determine radioactivity released from the tissue, an aliquot of each fraction (0.5 ml) was also assayed for radioactivity. The radioactivity released from the tissue in response to electrical stimulation has been shown previously to be [3H]NA by separation from its metabolites with high-performance liquid chromatography followed by radiochemical detection.22 The release of tritium was expressed as a fractional rate, i.e. as a percentage of the amount of radioactivity in the tissue at the time the release was determined. Resting release was calculated, unless stated otherwise, as the average of the fractional resting release (FRR) measured over two collection periods, in the sixth and seventh collection periods (R1), and compared with release measured in the 11th and 12th collection periods (R2) (fractional resting release 2 minus fractional resting release 1 (FRR2"FRR1)=ÄFRR). Nicotinic agonists were added from the 10th to 19th collection periods and the ability to increase the resting release of [3H]NA was measured. Antagonist affinity was expressed as the apparent dissociation constant (KD) calculated from the equation of Gaddum:7 KD=(A)/(DR"1), where (A) is the concentration of the antagonist and DR is the agonist dose ratio produced by the antagonist. Antagonists were generally tested at one concentration. The agonist 50 value is the concentration of the agonist that elicits 50% of the maximum effect and was calculated by Dose–Effect Analysis (Biosoft). Statistical analysis All data in the text are expressed as means&S.E.M. The statistical significance of the results was determined using analysis of variance (ANOVA) followed by Dunn’s test; P<0.05 was considered significant. Materials The following drugs were used: (")-nicotine, mecamylamine, (+)-tubocurarine, hexamethonium, á-bungarotoxin, (+)-anatoxin-a, (")-lobeline, dihydro-âerythroidine (DHâE), á-conotoxin, conotoxin GVIA and cytisine, obtained from Research Biochemicals International (Natick, MA, U.S.A.) or Sigma Chemical Co. (St Louis, MO, U.S.A.). á-Conotoxin-IMI was a gift from Dr J. M. McIntosh (University of Utah). (")-Epibatidine was synthesized by Dr Cs. Sza´ntay (Central Institute of Chemistry, Hungarian Academy of Sciences, Budapest, Hungary) and nbungarotoxin was a gift from Dr Liang (Jiangxi Medical College, P. R. China). RESULTS

Uptake and release of [3H]noradrenaline in the hippocampus and subregions (CA1, CA3 and dentate gyrus): effect of dimethylphenylpiperazinium The uptake of [3H]NA varied regionally. Uptake, after a 45-min incubation with [3H]NA, was highest in the DG and lowest in the CA1 region of the hippocampus (Table 1). The fractional release of radioactivity during 14 3-min resting periods was higher in the CA1 region. DMPP, a nicotinic agonist, increased the resting evoked release of [3H]NA in the CA1, CA3 and DG regions of the hippocampus and whole hippocampal

Nicotine receptors involved in hippocampal noradrenaline release Table 1. Uptake and release of [3H]noradrenaline in the hippocampus and subregions, CA1, CA3 and dentate gyrus

Region Hippocampus CA1 CA3 DG

Uptake (fmol/g tissue) (n=10)

Fractional release (%) of [3H]NA† (n=10)

960&52 805&120 981&91 1203&95*

0.57&0.02 0.70&0.02** 0.54&0.01 0.55&0.02

Uptake of [3H]NA after a 45-min incubation, expressed as fmol/g tissue. *P<0.05, DG significantly different from CA1. †Release measured during the 3-min collection periods. **P<0.001 versus hippocampus, CA3 and DG.

slices; the effects were concentration dependent (Fig. 1). When the effect of different concentrations of DMPP was plotted against [3H]NA release, it resulted in a bell-shaped curve; for example, in whole slices and the DG, at the higher concentrations DMPP released less [3H]NA than at lower ones. There were no large differences in the release and the effect of DMPP to release [3H]NA in the three subregions of the hippocampus. From the curves, the apparent 50 values were approximately 6.8, 11.2, 7.5 and 11.8 µM for CA1, CA3, DG and for the whole slice, respectively. The subtle differences in the dose–response profiles between the three regions may reflect changes in efficacy of [3H]NA uptake across different experiments. Further studies may be needed to explore this possibility. Nevertheless, in subsequent experiments hippocampal slices were used that involved all the three subregions. In this preparation the fractional release at rest was 0.57&0.02% (n=10; Table 1). The effect of different nAChR agonists to release [3H]NA is shown in Fig. 2. nAChR agonists released NA in a concentration-dependent manner with differing efficacies and potencies. Since the maximum releasing effect (Emax values, Table 2) produced by the agonists was different, normalized values were also plotted in Fig. 2. In addition to DMPP, (")nicotine, (+)-anatoxin-a, a potent stereospecific nAChR agonist,41 (")-epibatidine, an alkaloid with nAChR agonist activity,28 (")-lobeline and cytisine released NA. Concentration–response studies revealed markedly different 50 values for [3H]NA release evoked by nAChR agonists. (")-Epibatidine is a 500-fold more potent agonist than DMPP. The maximum effect (28&3% increase) produced by cytisine was significantly less, 31.5% of that produced by DMPP. While (")-epibatidine and (+)-anatoxin-a were potent in releasing [3H]NA, DMPP, cytisine, (")-nicotine and (")-lobeline were less potent in releasing [3H]NA. The maximum responses to epibatidine, anatoxin and cytisine were less than 50% of the maximum release (ÄFRR) evoked by DMPP and lobeline (Table 2, Fig. 2).

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The low efficacy of cystine was in agreement with electrophysiological studies.26 However, higher efficacies were expected with the other agonists, but this was not the case in our experiments. Epibatidine and (+)-anatoxin-a were slightly higher, and the other agonists two- to five-fold higher in efficacies than cytisine. Although not measured, these differences could possibly be accounted for by differences in desensitization developing even during the 3-min collection period. The bell-shaped concentration– response curves seem likely to be due to the desensitization of nAChR channels, or even an inhibition of the channel produced by long-lasting exposure to a high concentration of agonist. In the case of DMPP, the desensitization occurred only after two 3-min collection periods (Fig. 3). Figure 3 shows the concentration-dependent effect of DMPP on the fractional release (%) of [3H]NA, obtained in a different series of experiments. The effect of DMPP on [3H]NA release was not maintained, although DMPP was present in the perfusion fluid until the end of the experiment. The decrease in response with time observed in the effect of DMPP did not depend on the concentration of applied DMPP.

Effect of calcium channel antagonists, tetrodotoxin and nicotinic antagonists on dimethylphenylpiperaziniuminduced [3H]noradrenaline release When Ca2+ was removed and EGTA (1 mM) was added to the Krebs solution, the effect of DMPP on [3H]NA was significantly reduced (Table 3). To determine whether the DMPP-induced NA release might be related to an action on calcium channels and, if so, which channel subtype(s) might be involved, we tested the ability of a series of calcium channel antagonists to block or attenuate DMPPinduced release of [3H]NA. The L-type channel blocker, nifedipine, had no effect on [3H]NA release induced by DMPP. The N-type channel blockers, Cd2+ and ù-conotoxin, attenuated the release induced by DMPP. In the absence of DMPP, Cd2+ had no effect on resting release (Table 3). The DMPP-induced release of [3H]NA was tetrodotoxin sensitive. á-Conotoxin-IMI, an nAChR antagonist selective for the á7 subunit (J. McIntosh, personal communication), also had no effect on DMPP-induced release. To assess whether the DMPP-induced release of [3H]NA was mediated by nAChRs and to make an attempt to pharmacologically characterize the subunit composition of nAChRs involved in this releasing action, the sensitivity of this effect to a series of antagonists was also tested (Table 3), and the apparent dissociation constants (KD) were calculated (method of Gaddum7). The nicotinic antagonists n-bungarotoxin, mecamylamine, hexamethonium and (+)-tubocurarine by themselves did not affect the resting release, but they antagonized the release of

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Fig. 1. The effect of DMPP on the release of [3H]NA from three different subregions (CA1, CA3 and DG) and whole hippocampal slices at rest (Ä resting, %). Superfusion with Krebs solution, at a rate of 0.5 ml/min, 5% CO2+95% O2, at 37)C. The total amount of [3H]NA above resting release was calculated (Ä resting, %) and was plotted against the concentration of DMPP. DMPP was added at the 10th collection period. Resting release was calculated as the average of the release measured in the sixth and seventh collection periods (FRR1), and compared with release measured in the 11th and 12th periods (FRR2); Ä resting=FRR2"FRR1. Results are the mean&S.E.M. (where given); n=3–5. The curves were fitted to a third-order regression analysis (Sigma plot, Jandel Scientific).

NA evoked by 20 mM DMPP. The calculated apparent dissociation constants (KD; µM) were 0.03&0.003 for n-bungarotoxin, 0.98&0.04 for mecamylamine, 6.05&0.84 for (+)-tubocurarine and 13.17&2.54 for hexamethonium (n=4 or 5). The apparent dissociation constants calculated show that n-bungarotoxin is the most effective antagonist. By contrast, á-bungarotoxin and DHâE, at concentrations up to 10 µM, had no effect.

DISCUSSION

It is now widely accepted that one of the main roles for neuronal nAChRs is to modulate the release of other neurotransmitters. The pharmacological characterization of the different physiological receptors is an important issue toward the elucidation of the role of nAChRs, in addition to a mode of action on to a specific nAChR subtype. In situ hybridization and receptor binding studies (cf. Ref. 21) suggest that more than one variant of á and â subunit combinations exists in the CNS. The evidence in support of different subtypes of nicotinic receptors, with functional significance, has been

obtained mainly from direct ligand-binding studies, and more recently by means of molecular, immunological and electrophysiological techniques (cf. Refs 21 and 37). It has been shown21,37 that neuronal nAChRs are clearly distinct from muscle nAChRs and are themselves diverse, even in the hippocampus.1 Electrophysiological evidence was obtained that inhibitory and excitatory presynaptic receptors play important roles in hippocampal function.41 Measuring the release of [3H]NA from the hippocampus and studying the effect of different nicotinic agonists and antagonists, conclusions can be drawn as to the nature of the presynaptic nAChRs located on the nerve terminals of noradrenergic fibers that are derived from the locus coeruleus.24 Freund et al.6 suggested that nicotine exerts effects via a subclass of nAChRs present in the hippocampus that are neither neuromuscular nor ganglionic in the classical sense. Others have identified independent nicotinic and muscarinic responses, selectively blocked only by antagonists of that particular class,30,31 or mixedtype cholinergic receptors sensitive to both nicotinic and muscarinic ligands.40 Additionally, there are a large number of NA varicosities present in the

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Fig. 2. Dose–response curves for nicotinic agonists on release of [3H]NA. Nicotine agonists were added to the superfusion Krebs solution from the eighth to 19th collection periods. Upper panel shows the concentration-dependent effect of agonists on resting release (ÄFRR). Maximal release was normalized to 100% for each agonist (lower panel). See Table 2 for 50 and Emax values. Results are means&S.E.M.; n=4–8 replicates at four to six concentrations. Table 2. Release of [3H]noradrenaline by nicotinic acetylcholine receptor agonists from hippocampal slice preparations (50 and Emax values) Emax Agonist (")-Epibatidine (+)-Anatoxin-a DMPP Cytisine (")-Nicotine (")-Lobeline

50

ÄFRR

FRR2/FRR1

19.6&13.7 nM 1.77&6.6 µM 10.6&5.9 µM 22.5&17.3 µM 34.6&6.6 µM 71.0&9.5 µM

0.29&0.09 0.30&0.02 0.73&0.07 0.23&0.04 0.45&0.03 1.53&0.41

1.49&0.04 1.54&0.04 2.28&0.14 1.38&0.19 1.84&0.08 3.85&0.82

Nicotine agonists were added to the superfusion Krebs solution from the 10th to 19th collection periods. Effects on resting (FRR2/FRR1) and difference between FRR2 and FRR1 (ÄFRR) are given. Results are means&S.E.M.; n=4–8 replicates at four to six concentrations. 50 values were calculated by Dose–Effect Analysis (Biosoft).

hippocampus originating from the locus coeruleus;24 however, there have been few studies characterizing the presynaptic modulation of NA release. We therefore attempted to characterize the effect of nicotinic agonists and antagonists on the release of NA from the hippocampus. The highest uptake of [3H]NA was seen in the DG and the lowest in the CA1 region. The uptake agrees with the distribution of NA,22 NA varicosities observed by immunostaining,17 and with measure-

ments of the density of NA receptors.50 Nicotinic receptor agonists released [3H]NA in a (Ca2+)odependent manner from noradrenergic axon terminals in all three subregions of the hippocampus. The effect of nicotinic receptor agonists was concentration dependent. The rank order of potency to release NA was (")-epibatidine>(+)-anatoxin-a> DMPP>cytisine>(")-nicotine>(")-lobeline. Since DMPP is generally accepted as a nicotinic agonist, and the DMPP-induced release (Emax) was much

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Fig. 3. Time- and concentration-dependent effect of DMPP on the fractional release of [3H]NA from hippocampal slices. DMPP was added to the perfusion solution and kept in throughout the experiments, as indicated. Note that the releasing effect was transient; the effect of DMPP was not maintained (n=3–5).

greater than that seen with epibatidine, (+)anatoxin-a and cytisine, we further characterized its effect on [3H]NA release. In functional studies in the ganglion (a postsynaptic neuronal site), the rank order of potency of antagonists was mecamylamine>-tubocurarine> hexamethonium>DHâE. At the neuromuscular junction (a postsynaptic muscle site), the rank order of potency of antagonists is á-bungarotoxin>pancuronium>(+)-tubocurarine±hexamethonium. In our study, in which the presynaptic nAChRs located on noradrenergic axon terminals in the hippocampus were tested, n-bungarotoxin> mecamylamine>(+)-tubocurarine>hexamethonium± á-bungarotoxin=DHâE was the order of potency. The finding that á-bungarotoxin was completely ineffective excludes the possibility that nAChRs involved in the release of NA contain á7 subunits, one of the predominant subtypes of nAChRs in the hippocampus, and one involved in long-term potentiation.15 This is confirmed by the lack of effect of á-conotoxin-IMI, an nAChR antagonist selective towards the á7 subunit (J. McIntosh, personal communication). Agonists have also been useful in distinguishing muscle nAChRs from neuronal ones and in distinguishing between different neuronal nAChRs. Mulle et al.23 found that the rank order of potency of agonists for neurons from rat interpeduncular nucleus was cytisine>ACh>nicotine; for nAChRs on neurons from the medial habenula, it was nicotine>cytisine>ACh. In our experiments, (")-epibatidine was about 500–3500 times more potent an agonist than the others (Fig. 2, Table 1), and (+)-anatoxin-a>DMPP>cytisine>(")-nicotine> (")-lobeline was the rank order of potency. Sacaan et al.35 reported a similar rank order of potency for three agonists, DMPP>(")-cystine>(")-nicotine, for evoking [3H]NA release. These differences may be explained by differences in nAChR subunit composi-

tion.18 It is cautioned that the studies of Luetje and Patrick18 showed only partial dose–response profiles, which failed to use high enough doses of agonists to achieve maximum cell currents, thereby precluding evaluation of absolute efficacies of agonists. The present study examined full dose–response profiles, and also based the subunit assignment on the antagonism by n-bungarotoxin. n-Bungarotoxin has selectivity towards the á3â2 subunit combination, but does not block á3â4 combinations.2 The pharmacological profiles in the present study, showing an n-bungarotoxin block and no á-bungarotoxin block, provide strong support for the involvement of nAChR á3 and â2 nAChR subunits in the control of NA release from hippocampal slices. Therefore, it is possible that the functional nicotinic (DMPP) responses of noradrenergic axon terminals of the hippocampus correspond to an nAChR consisting of á3 and â2 subunits. However, interpretation of the subunit composition of native receptors, based on pharmacological evaluations carried out in Xenopus oocytes, should be viewed with caution. In many cases electrophysiological studies of brain nicotinic receptors have failed to show good correlations with oocyte data.23 Additionally, the subtype selectivity of n-bungarotoxin is complex; some subtypes show inhibition to n-bungarotoxin, but with rapid kinetics of onset and recovery (á3â4), in contrast to the slow and prolonged inhibition of á3â2-containing receptors.25 While the á3â2 subtype combination is not very abundant in human brain,33 in rats â2 is the most widely expressed â subunit.49 The á3 mRNA is also present in monkey hippocampus,4 while scarcely labeled in human brain.33,34 It is important to recognize that the nicotinic subunits of nAChRs on NA terminals will be synthesized in the cell bodies, i.e. in the locus coeruleus, where expression of mRNA should be examined. Wada et al.49 found high levels of á3 and â2 mRNA expressed there, with little á4. This composition of presynaptic nAChRs is different from that of somatodendritic nAChRs,48 in which the receptor is composed of á4â2 subunits. The present study does not exclude the involvement of the á4â2 subtype. However, this possibility is not favored by the finding that DHâE failed to exert an antagonistic effect, DHâE generally having a high selectivity to the á4â2 receptor subtype. Also, the affinity of the nicotinic agonist, cytisine, has been assigned to the á4â2 and á7 subunits. Since they also released NA, but with different efficacy and affinities, it is possible that multiple subunits are involved in the presynaptic release of NA, of which the DMPP response involves the á3â2 subunit. Such assignments should also be independently supported by protein chemical and/or molecular biological evidence before such a definitive conclusion is drawn. The wide variety of subunit composition of nAChRs is not surprising, since it has been shown44 that neurons may express nAChRs composed of more than

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Table 3. Effect of Ca2+ removal, calcium channel antagonists, tetrodotoxin and nicotinic antagonists on the release of [3H]noradrenaline [3H]NA release Resting† Drugs and/or conditions Control (11) Ca2+ removal+EGTA, 1 mM (6) Nifedipine, 50 µM (5) Cd2+, 125 µM (5) ù-Conotoxin, 0.1 µM (5) Tetrodotoxin, 1 µM (10) n-Bungarotoxin, 0.5 µM (4) á-Bungarotoxin, 10 µM (4) Mecamylamine, 10 µM (4) Pancuronium, 100 µM (4) -Tubocurarine, 100 µM (4) Hexamethonium, 300 µM (4) DHâE, 10 µM (4) á-Conotoxin-IMI, 40 nM (4)

Evoked by DMPP (20 µM)‡

FRR2/FRR1

ÄFRR

FRR2/FRR1

ÄFRR

0.95&0.02 0.90&0.02 0.94&0.07 0.90&0.02 1.02&0.02 0.95&0.03 1.00&0.02 0.94&0.06 0.89&0.06 0.96&0.02 0.94&0.05 0.96&0.02 0.95&0.03 0.95&0.02

"0.02&0.01 "0.05&0.02 "0.04&0.02 "0.06&0.09 0.02&0.01 "0.03&0.02 0.03&0.01 "0.02&0.04 "0.06&0.05 "0.03&0.02 "0.04&0.03 "0.02&0.01 "0.03&0.02 "0.02&0.01

1.73&0.07* 1.22&0.03** 1.81&0.09 1.29&0.09** 1.16&0.04** 1.23&0.07** 1.09&0.03** 2.07&0.09 1.12&0.03** 1.27&0.05** 1.23&0.04** 1.14&0.04** 1.89&0.07 1.62&0.11

0.38&0.03* 0.15&0.02** 0.49&0.08 0.25&0.03** 0.08&0.02** 0.09&0.02** 0.05&0.02** 0.55&0.07 0.07&0.02** 0.16&0.02** 0.15&0.03** 0.08&0.02** 0.50&0.04 0.34&0.06

Fractional resting release (FRR) represents the amount of [3H]NA release as a percentage of the total tissue radioactivity present at the time of measurement. Drugs were added or the conditions were introduced to the perfusion Krebs solution at the fifth collection period (calcium removal or calcium antagonist added) or the eighth collection period for the other drugs, and maintained throughout the experiments. †The fractional release of [3H]NA measured during the ninth and 10th collection periods was compared to that measured in the first and second collection periods (FRR2/FRR1 and ÄFRR=FRR2"FRR1). ‡The fractional release of [3H]NA evoked by DMPP (20 µM) during the 11th and 12th collection periods was compared to radioactivity released in collection periods 9 and 10. DMPP was added to the perfusion fluid at the 10th collection period till the end. *P<0.05 in comparison with the non-treated control, **P<0.05 in comparison with the control. ANOVA followed by Dunn test was used for statistical analysis. Results are mean&S.E.M. (number of experiments is indicated in parentheses).

two types of subunits, and more than one nAChR subtype. Release of NA evoked by nAChR stimulation (DMPP) was external Ca2+ dependent (Table 3), and it was found that Cd2+, a cation that blocks both L- and N-type Ca2+ channels, reduced the release, but nifedipine, an L-type Ca2+ channel blocker, had no effect. The calcium N-type channel antagonist, ù-conotoxin-GVIA (100 nM), but not nifedipine, inhibited the release of NA evoked by nAChR stimulation. These findings suggest that N-type voltagesensitive Ca2+ channels are involved in release of NA evoked by nAChR stimulation. In CNS neurons, the involvement of L- or N-type channels in transmitter release appears to depend on the stimulus used to evoke release. It has been shown that N-type channels play a dominant, if not exclusive, role in controlling NA release, which is dihydropyridine resistant, but sensitive to Cd2+ and ù-conotoxin.13 Nicotinic receptor (DMPP)-mediated release of NA in human neuroblastoma SH-SY5Y cells is activated by L-type calcium channels.43 In our experiments about 30% of NA released by DMPP was not antagonized by one of the calcium channel blockers (ù-conotoxin) applied. It seems likely that a part of the release was due to calcium influx through the nAChR-operated channels, and another part related to calcium influx through voltage-dependent N-type channels. This

may account for the observed tetrodotoxin sensitivity. Activation of nicotinic channels, with resulting Ca2+ and Na+ influx via these channels, can produce sufficient local depolarization and generation of action potentials at the preterminal areas, which are able to open voltage-sensitive calcium channels, channels sensitive to conotoxin and Cd2+, and tetrodotoxin. Alternatively, since tetrodotoxin has not been shown to be active at any nAChR subtype tested to date, it raises the possibility that these nicotinic receptors are located on interneurons in the slices. CONCLUSION

Since the hippocampus receives a noradrenergic innervation from the locus coeruleus and NA inhibits excitatory synaptic transmission in area CA3,38,39,42 and there is a dense cholinergic projection from the medial septum/diagonal band,16 the question arises as to the functional role of this nAChR-mediated, NA-releasing action of cholinergic innervation described in this paper. The effectiveness of this interaction is supplemented by the fact that the noradrenergic axon terminals are not equipped with inhibitory muscarinic receptors and the release of NA is not subjected to muscarinic receptor-mediated inhibition.22 This means that excessive firing of cholinergic afferents or nicotine may produce an

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Fig. 4. Presynaptic interactions in the hippocampus. Norepinephrine (NE) released either by axonal firing or by the stimulation of nAChRs (by (")-nicotine for example) may be able to inhibit (via stimulation of á1-adrenoceptors39) the release of glutamate (Glu) from excitatory mossy fibers and recurrent collaterals; thus, the stimulation of nAChRs located on the noradrenergic axon terminals would result in a reduction of the activity of pyramidal cells. In addition, the noradrenergic axon terminals are equipped with N-methyl--aspartate and non-N-methyl--aspartate receptors able to release norepinephrine. These data support the hypothesis that the net effect of nAChR stimulation on pyramidal cells is a complex function of local circuit interactions.

increase of NA release not associated with neuronal firing. The tonic release of NA may result in an excess of NA, which is able to reach remote target cells and axon terminals,45,47,48 and exert modulatory action. The excessive release of NA evoked by nAChR stimulation in the hippocampus may result in a decrease of the evoked release of transmitter from excitatory terminals of both mossy fibers and CA3 pyramidal cell recurrent collaterals39 (Fig. 4). NA released in response to cholinergic signals or nicotine

in this manner is proposed to set the background, steady-state level of extraneuronal NA in the hippocampus. Acknowledgements—This work was supported by a grant from the Council for Tobacco Research U.S.A., Inc., the Hungarian Research Fund (OTKA), the Medical Research Council (ETT) and a grant (CIPA-CT92-3014) for Cooperation in Science and Technology with Central and Eastern European Community. The authors also thank Dr Ron Lukas for his constructive comments.

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