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NEUROPHARMACOLOGY

Nicotinic Acetylcholine Receptor-Mediated [³H]Dopamine Release from Hippocampus

Neurological Diseases Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois

Received August 27, 2004; accepted November 12, 2004.

	Abstract

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The mechanism of nicotinic acetylcholine receptor (nAChR)-induced hippocampal dopamine (DA) release was investigated using rat hippocampal slices. nAChRs involved in hippocampal DA and norepinephrine (NE) release were investigated using prototypical agonists and antagonists and several relatively novel compounds: ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine], (±)-UB-165 [(2-chloro-5-pyridyl)-9-azabicyclo [4.2.1]non2-ene], and MG 624 [N,N,N-triethyl-2-[4-(2 phenylethenyl)phenoxy]-ethanaminium iodine]. (±)-Epibatidine, (±)-UB-165, anatoxin-a, ABT-594, (-)-nicotine, 1,1-dimethyl-4-phenyl-piperazinium iodide, and (-)-cytisine (in decreasing order of potency) evoked [³H]DA release in a mecamylamine-sensitive manner. Aside from (±)-UB-165, all the agonists displayed full efficacy relative to 100 µM (-)-nicotine in [³H]DA release. In contrast, (±)-UB-165 was a partial agonist, evoking 58% of 100 µM (-)-nicotine response. Mecamylamine, MG 624, hexamethonium, d-tubocurare, and dihydro--erythroidine (in decreasing order of potency), but not -conotoxin-MII, methyllycaconitine, -conotoxin-ImI, or -bungarotoxin, attenuated 100 µM (-)-nicotine-evoked [³H]DA release in a concentration-dependent manner. (±)-UB-165, ABT-594, and MG 624 exhibited different pharmacologic profiles in the [³H]NE release assay when compared with their effect on [³H]DA release. ABT-594 was 4.5-fold more potent, and (±)-UB-165 was a full agonist in contrast to its partial agonism in [³H]DA release. MG 624 potently and completely blocked NE release evoked by 100 µM (-)-nicotine and 10 µM (±)-UB-165, whereas it only partially inhibited (-)-nicotine-evoked [³H]DA release. In conclusion, we provide evidence that [³H]DA can be evoked from the hippocampus and that the pharmacologic profile for nAChR-evoked hippocampal [³H]DA release suggests the involvement of 34^* and at least one other nAChR subtype, thus distinguishing it from that of nAChR-evoked hippocampal [³H]NE release.

A major contributor to cognitive function seems to be the neurotransmitter DA. Although precise contribution of DA remains unclear, it most likely plays a role in the regulation of several different aspects of cognitive brain function (Nieoullon, 2002). Abnormal dopaminergic neurotransmission has been observed in several diseases and disorders that express cognitive dysfunctions, including Alzheimer's disease, Tourette's disease, Huntington's chorea, attention deficit hyperactivity disorder, and cognitive deficits associated with schizophrenia.

In the hippocampus, a critical area involved in attention and memory, the significance of nicotinic-dopaminergic interactions for cognitive function has been well documented (Levin and Simon, 1998) and includes evidence of a significant role for nicotinic receptors localized in the ventral hippocampus in working memory. Projections from the mesencephalic DA groups [ventral tegmental area-substantia nigra (VTA-SN)] to the hippocampus have been characterized, with the most innervated area being the ventral region of the hippocampal formation (Jay, 2003). DA (Jay, 2003), the DA transporter (Horn, 1990; Mennicken et al., 1992), and DA receptors (Jay, 2003) also have been detected in rat hippocampus, substantiating dopaminergic function. Moreover, DA influences the firing rate of hippocampal cells (Smialowski and Bijak, 1987) and plays a role in cognitive function in the hippocampus (Jay, 2003), particularly with respect to working memory. In vivo microdialysis studies have demonstrated that behaviorally active doses of nicotine increase hippocampal DA release (Brazell et al., 1991). Moreover, dopaminergic neurons of the VTA-SN express mRNA for a number of nAChR subunits, including 3 through 7 and 2 through 4 (Charpantier et al., 1998; Klink et al., 2001; Azam et al., 2002). To date, the nAChR subtype(s) mediating hippocampal [³H]DA release have not been characterized. Therefore, using several different agonists and antagonists, the present study used our relatively novel method of measuring neurotransmitter release (Anderson et al., 2000; Puttfarcken et al., 2000) to investigate nAChR-evoked hippocampal [³H]DA release from the hippocampus. The potential involvement of 42 and 7 was investigated because previous behavioral studies have demonstrated the involvement of these subtypes with working memory function in the hippocampus (Levin and Simon, 1998). Furthermore, the role of 34 was examined by comparing results obtained for [³H]DA release with those obtained for [³H]NE release from the hippocampus, a process reported to be primarily mediated by 34 (Clarke and Reuben, 1996; Wonnacott, 1997; Luo et al., 1998; Anderson et al., 2000).

To our knowledge, this is the first study to extensively characterize nAChR-mediated [³H]DA release from the hippocampus. We report on the effects of ABT-594, a compound with high affinity for the 42 subtype that also is a potent agonist at 42 and (albeit to a somewhat lesser extent) 34 receptor in rats and humans (Donnelly-Roberts et al., 1998). Furthermore, we report for the first time the pharmacologic actions of two novel compounds, MG 624 and (±)-UB-165, in the hippocampus. Although little is known regarding its selectivity for specific nAChRs in the rodent brain, MG 624 has been reported to display nanomolar affinity and antagonist activity against rat 7 receptors expressed in Xenopus oocytes (Di Angelantonio et al., 2000). (±)-UB-165, a hybrid of anatoxin-a and epibatidine, has been demonstrated to functionally activate recombinant rat 34 expressed in a mouse fibroblast cell (Sharples et al., 2002).

	Materials and Methods

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Materials. [³H]DA (3,4-[ring-2,5,6-³H]-dihydroxyphenylethylamine, 60 Ci/mmol) and [³H]NE (1-[ring-2,5,6-³H]-norepinephrine) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). (-)-Nicotine bitartrate, (+)-anatoxin-a, 1,1 dimethyl-4-phenyl-pipazinium iodide (DMPP), (-)-cytisine, hexamethonium (Hex) bromide, d-tubocurare (d-TC), mecamylamine (Mec), dihydro--erythroidine HBr (DHE), methyllycaconitine (MLA), desipramine, pargyline, and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO). (±)-Epibatidine (EB) and nomifensine were purchased from Sigma/RBI (Natick, MA). -Conotoxin MII (-CtxMII) was obtained from Tocris Cookson Inc. (Bristol, UK). (±)-UB-165 fumarate and MG 624 were obtained from Tocris Cookson Inc. -Conotoxin ImI (-CtxImI) was purchased from either American Peptide Co., Inc. (Sunnyvale, CA) or Sigma-Aldrich. ABT-594 was synthesized in house.

Animals. Male Sprague-Dawley rats (250-300 g) (Harlan, Indianapolis, IN) were housed four per cage, and food and water were available ad libitum. Rats were allowed to acclimate to housing conditions 4 days after arrival. Animals were treated in accordance with the Institutional Animal Care and Use Committee guidelines.

Measurement of [³H]DA and [³H]NE Release from Tissue Slices. Methods for tissue slice preparation and measurement of [³H]DA release were slightly modified from those described elsewhere (Puttfarcken et al., 2000). Briefly, rat hippocampal slices (250 µm) were prepared using a McIlwain tissue chopper. The tissue was washed three times with buffer [15 mM HEPES-NaOH, 137 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO₄, 0.1 mM ascorbic acid, 2.5 mM CaCl₂, 1.25 mM NaH₂PO₄, 3.3 mM dextrose, and 10 µM pargyline (pH 7.4)] and subsequently incubated with buffer containing uptake blockers for 5 min at 37°C under an atmosphere of 95% O₂ and 5% CO₂. Tissue was then loaded with either 100 nM [³H]DA or 50 nM [³H]NE for 30 min. The present study used a relatively low concentration of [³H]DA (100 nM) to ensure exclusive uptake by the DA transporter because DA exhibits a weak affinity for the NE and serotonin transporter (K_D = 32 and >100 µM, respectively) (Tatsumi et al., 1997). Desipramine (100 nM) was included in the assay buffer during the loading of [³H]DA to prevent uptake into noradrenergic terminals. In experiments examining the actions of -bungarotoxin (-BTX), -CtxMII, and -CtxImI, tissue was allowed to incubate at 37°C in buffer containing 0.1% bovine serum albumin and 1 mM phenylmethylsulfonyl fluoride. After incubation, the tissue suspension was allowed to settle under gravity and washed extensively to remove excess radioactivity. The slices were distributed in 50 µl/well to 96-well plates containing a 1.2-µm hydrophilic, low protein-binding Durapore membrane (MultiScreen BV clear plates, catalog no. MABVN1210; Millipore Corporation, Billerica, MA) and washed three times with 100 µl buffer/well using vacuum filtration (Polyfiltronics UniVac; Whatman Inc., Clifton, NJ). Hippocampi from two animals were pooled to provide enough tissue for one 96-well plate. After a 10-min rest period at 37°C, the buffer was removed by vacuum filtration. For experiments involving -BTX, slices were distributed to 96-well plates in the presence of the antagonist and allowed to incubate at 37°C for 60 min. After incubation, the buffer then was removed by vacuum filtration. For the [³H]DA release assay, the subsequent buffer contained 100 nM nomifensine; for the [³H]NE release assay, 1 µM yohimbine and 30 nM nisoxetine were added. To collect the basal release, 100 µl of preoxygenated buffer was added to each well and allowed to preincubate for 5 min at 37°C. Preincubation was terminated by vacuum filtration into a 96-well plate to collect basal release. After collection of the basal sample, 100 µl of buffer containing various concentrations of agonists (with or without antagonists) were added to each well and allowed to incubate for an additional 5 min at 37°C. Stimulated release was collected by rapid filtration into a new 96-well collecting plate. After the collection of stimulated release, tissue samples were collected and counted for radioactivity as described previously.

Data Analysis. Data are presented as fractional release for each well [stimulated release/(radioactivity in stimulated + tissue lysate) - basal release/(radioactivity in basal + stimulated + tissue lysate)] and expressed as mean ± S.E.M. Relative efficacies were calculated using the release evoked by 100 µM (-)-nicotine as a standard. EC₅₀ and IC₅₀ values were determined by fitting the data to a sigmoidal logistic equation using the software Prism (GraphPad Software Inc., San Diego, CA).

	Results

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The Effect of nAChR Agonists on Hippocampal [³H]DA Release. Activation of nAChRs evoked [³H]DA release in the rat hippocampus. The nAChR agonists ABT-594, (±)-UB-165 (Fig. 1A), EB, DMPP, anatoxin-a, and (-)-cytisine (Fig. 1B) evoked a concentration-dependent increase in [³H]DA release, with a rank order potency of EB > (±)-UB-165 > anatoxin-a > ABT-594 > (-)-nicotine > DMPP > (-)-cytisine (Table 1). With the exception of DMPP, pretreatment with Mec completely blocked release evoked by all the agonists tested (Table 1). Mec only inhibited 48% of the release evoked by DMPP (Table 1). (-)-Cytisine was the weakest agonist examined, with an EC₅₀ value 3.4-fold less potent than that obtained for (-)-nicotine (Fig. 1B; Table 1). The maximal release produced by all the agonists, aside from (±)-UB-165, was equivalent to that produced by (-)-nicotine (Table 1). In contrast, (±)-UB-165, the hybrid of anatoxin-a and epibatidine (Sharples et al., 2000), produced only 58% of the release evoked by (-)-nicotine (Fig. 1A; Table 1).

View larger version (29K): [in this window] [in a new window]   Fig. 1. Concentration-response curves of agonist-induced [3H]DA and [3H]NE release from rat hippocampal slices. Rat hippocampal slices were preloaded with either [3H]DA (A and B) or [3H]NE (A) and exposed to different agonists, as described under Materials and Methods. Results were normalized and expressed as a percentage of 100 µM (-)-nicotine-induced release. Data points represent the mean ± S.E.M. of four to five separate experiments performed in quadruplicate.

The Effect of (±)-UB-165 and ABT-594 on Hippocampal [³H]NE Release. The ability of ABT-594 and (±)-UB-165 to evoke [³H]NE release from hippocampus also was examined for further characterization of the nAChR subtypes involved in (-)-nicotine-evoked [³H]NE release (Fig. 1A, Table 1). Previous studies of ours (Anderson et al., 2000) have already examined the other agonists used in the present study. Both agonists evoked a concentration-dependent increase in [³H]NE release, with EC₅₀ values of 0.47 µM for ABT-594 and 0.058 µM for (±)-UB-165. Pretreatment with Mec completely blocked release evoked by both agonists (Table 1). The maximal release produced by ABT-594 and (±)-UB-165 was nearly equivalent to that produced by (-)-nicotine (Table 1).

The Effect of nAChR Antagonists on Hippocampal [³H]DA Release. Additional experiments examined the ability of various antagonists to affect hippocampal [³H]DA release evoked by (-)-nicotine (Figs. 2, 3, 4; Table 2). A maximal concentration of (-)-nicotine (100 µM) was used in these experiments. Most of the antagonists tested inhibited (-)-nicotine-evoked release, with a rank order of potency of Mec > MG 624 > Hex > d-TC > DHE (Fig. 2; Table 2). The weakest antagonist tested, DHE, only partially (by 67 ± 10%) inhibited the release (Fig. 2A; Table 2). In contrast to the antagonists mentioned above, three of the four reported 7 antagonists (MLA, -CtxImI, and -BTX) examined had no effect on 100 µM (-)-nicotine-evoked [³H]DA release (Fig. 3, A-C). Interestingly, MG 624, the fourth 7 antagonist, originally characterized in chicks (Gotti et al., 1998), blocked 60% of (-)-nicotine-evoked [³H]DA release (Fig. 2B; Table 2) and was one of the most potent compounds examined (IC₅₀ = 0.54 µM). In contrast, -CtxMII, the 323/623 antagonist, had no activity against [³H]DA release from the hippocampus (Fig. 3D).

View larger version (25K): [in this window] [in a new window]   Fig. 2. Concentration-inhibition curves of (-)-nicotine-induced [3H]DA (A and B) and [3H]NE (B) release from rat hippocampus by antagonists. Preloaded rat hippocampal slices were preincubated for 5 min with the indicated concentrations of antagonist before the addition of 100 µM (-)-nicotine. Treatment with antagonists alone had no effect on basal levels. Data were normalized to release evoked by 100 µM (-)-nicotine in the absence of antagonist. Data points represent the mean ± S.E.M. of five to six separate experiments performed in quadruplicate.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Concentration-inhibition curves of (-)-nicotine-induced [3H]DA release from rat hippocampus by MLA (A), -CtxIMI (B), -BTX (C), and -CtxMII (D). Basal release was collected after a 5-min preincubation with the indicated concentrations of antagonist before the addition of 100 µM (-)-nicotine. In -BTX experiments, slices were incubated for 60 min before the collection of basal samples. Treatment with antagonists alone had no effect on basal DA levels. Data were normalized to release evoked by 100 µM (-)-nicotine in the absence of antagonist (indicated by dotted line). Data points represent the mean ± S.E.M. of five separate experiments performed in quadruplicate.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effect of Mec (A), Hex (B), DHE (C), and MG 624 (D) on (-)-nicotine-induced [3H]DA release. Basal release was collected after a 5-min preincubation with the indicated concentrations of antagonist before the exposure of different concentrations of (-)-nicotine. Data were normalized to release evoked by 100 µM (-)-nicotine in the absence of antagonist. Data points represent the mean ± S.E.M. of three or four separate experiments performed in quadruplicate.

To obtain an idea of the mechanism underlying antagonism, (-)-nicotine concentration-response curves were carried out in the absence and presence of each antagonist (Fig. 4). Antagonist concentrations were selected based on IC₅₀ values obtained in earlier experiments (Table 2). The inhibition produced by each antagonist was found to be insurmountable, suggesting a noncompetitive mechanism.

The Effect of MG 624 on Hippocampal [³H]NE Release. Because the effect of MG 624, a reported 7 receptor antagonist, on nicotine-induced [³H]DA release was different from that of other 7 receptor antagonists, further experiments examined its ability to affect other nAChR-mediated neurotransmitter release processes. A maximal concentration of 100 µM (-)-nicotine (Fig. 2B; Table 2) and 10 µM (±)-UB-165 (Table 2) was used to evoke hippocampal [³H]NE release. In contrast to hippocampal [³H]DA release, MG 624 completely inhibited [³H]NE release evoked by (-)-nicotine and (±)-UB-165 with IC₅₀ values of 0.34 and 0.42 µM, respectively.

	Discussion

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To characterize nAChR(s) subtypes involved in (-)-nicotine-evoked [³H]DA release, we examined the ability of nAChR agonists to evoke [³H]DA release in rat hippocampus. Each agonist elicited a concentration-dependent release of hippocampal [³H]DA with a rank order potency somewhat similar to that for hippocampal [³H]NE release. This suggests that at least one of the same nAChR subtypes is involved in mediating release of both neurotransmitters from the hippocampus. Because previous studies have proposed that 34 is a major nAChR subtype involved in hippocampal [³H]NE release (Clarke and Reuben, 1996; Wonnacott, 1997; Luo et al., 1998; Anderson et al., 2000), the current data suggest that 34 also may be involved in hippocampal [³H]DA release. In support of this, (-)-cytisine displayed full agonism, suggesting involvement of 4 rather than 2 nAChRs (Luetje and Patrick, 1991; Papke and Heinemann, 1994). The rank order potency for (-)-cytisine also was similar for hippocampal [³H]DA release to that measured using 34 human recombinant nAChRs (Stauderman et al., 1998). Although DMPP seemed much more efficacious in evoking [³H]NE release (178%; Anderson et al., 2000), Mec did not fully block release for either neurotransmitter. A non-nicotinic, Ca²⁺-independent component of DMPP-mediated [³H]NE release has been reported previously (Kiss et al., 1997; Anderson et al., 2000). Because the concentration of nomifensine present in the assay is not expected to fully inhibit rat DA transporters (Richelson and Pfenning, 1984), DMPP could influence [³H]DA uptake, resulting in a carrier-mediated increase of extracellular DA. Finally, maximal DMPP responses equal to or greater than (-)-nicotine have been shown only with 3 subtypes (Luetje and Patrick, 1991).

Interestingly, despite similar agonist rank order potency for both neurotransmitters, the response to (±)-UB-165 (Sharples et al., 2000) and ABT-594 differed, suggesting distinct profiles of nAChRs involved in modulating release of each neurotransmitter. Although ABT-594 was equally efficacious in both assays, it was 4.5-fold more potent in the [³H]NE release assay. Furthermore, whereas (±)-UB-165 only partially evoked hippocampal [³H]DA release, it showed full efficacy in [³H]NE release, in agreement with efficacy reported for human (Sharples et al., 2000) and rat (Sharples et al., 2002) recombinant 34^* nAChRs. If the pharmacologic profile of (±)-UB-165 at rat recombinant and native nAChRs is similar, the partial efficacy of (±)-UB-165 suggests that other subtypes that are not significantly activated by (±)-UB-165 in humans, such as 42, 22, and/or an nAChR(s) composed of multiple and subunits, also may function in nAChR-mediated rat hippocampal [³H]DA release. Although the expression of the 2 nAChR subunit is weak to moderate in hippocampus (Wada et al., 1989), the weak antagonism by DHE does not support a role for 22 (IC₅₀ at human 22 = 850 nM) (Chavez-Noriega et al., 1997). Furthermore, 42^* is most likely not involved because the IC₅₀ value of DHE in the present this study is reminiscent of the weak antagonist activity displayed by DHE in two cell lines expressing 3 but not in 4-containing nAChRs (Decker et al., 1995; Puttfarcken et al., 1997). Although we have previously described DHE as a competitive antagonist for nAChR-mediated striatal [³H]DA release using our 96-well format (Anderson et al., 2000), and others have reported competitive properties for nAChR-mediated [³H]NE release from hippocampal synaptosomes (Clarke and Reuben, 1996) and [³H]DA release from rat striatal synaptosomes (Rapier et al., 1990; el-Bizri and Clarke, 1994), in hippocampus DHE behaved as a noncompetitive antagonist of [³H]DA release, as has also been reported for [³H]NE release (Anderson et al., 2000). Although the mechanism remains unclear, the apparent noncompetitive inhibition of hippocampal [³H]NE release (Anderson et al., 2000) by DHE was not attributable to differences in tissue preparation (slice versus synaptosomes), method of measuring release (superfusion versus 96-well format), or preincubation times (Anderson et al., 2000). Competitive antagonists can be classified into two different subtypes depending on the interaction between antagonist and receptor (Waud, 1968). The strength and/or mode of interaction between DHE and receptor may vary depending on the subunit composition, and DHE may behave as a nonequilibrium, or irreversible, competitive antagonist of specific nAChRs rather than as an equilibrium competitive antagonist. These types of antagonists show insurmountable competition, as also is typical of noncompetitive antagonists. The exact composition of nAChRs involved in hippocampal [³H]DA release is not yet fully defined, and the presence of a modulatory subunit, such as 5 (demonstrated by reverse transcription-polymerase chain reaction to be present in SN and VTA dopaminergic neurons) (Charpantier et al., 1998), may thus influence the properties of DHE. In support of this, 5 has been shown to alter nAChR sensitivity to different antagonists (Yu and Role, 1998).

The inability of three 7 antagonists, -CtxImI, MLA, and -BTX, to reverse (-)-nicotine-evoked [³H]DA release suggests that 7 is most likely not involved. However, the response of MG 624, a reported competitive antagonist at chick 7 receptors (Gotti et al., 1998; Maggi et al., 1999), differed between the two neurotransmitters. Although MG 624 was nearly equipotent, it only blocked 60% of nicotine-evoked [³H]DA release, whereas it fully antagonized nicotine- and UB-165-evoked [³H]NE release. Furthermore, in contrast to findings reported for the chick 7 receptor (Maggi et al., 1999), MG 624-mediated inhibition in the rat [³H]DA release assay seemed to be through a noncompetitive mechanism. This difference in the apparent mode of interaction may be because of different receptor subtype(s) involved and/or species-specific differences in the pharmacologic profile of oxystilbene derivatives (Di Angelantonio et al., 2000). Because 34 is thought to be a predominant nAChR involved in hippocampal [³H]NE release (Clarke and Reuben, 1996; Wonnacott, 1997; Luo et al., 1998; Anderson et al., 2000), these data suggest that MG 624 is an antagonist at 34^* nAChRs. Previous binding studies have shown that although oxystilbene derivatives, such as MG 624, retain their potency toward 7-containing receptors in mammals, they also are active at non-7-containing receptors (Gotti et al., 2000). In support of this, F3, a structurally related 4-oxystilbene derivative, competitively inhibits nicotine-evoked currents in a preparation that prevalently contains rat 34^* nAChRs (Di Angelantonio et al., 2000). The partial blockade of [³H]DA release by MG 624 suggests that other subtypes, aside from 34, also are involved in the release process. Results obtained with (±)-UB-165 further support the role of multiple nAChRs in the hippocampal [³H]DA release process.

The use of d-TC and Hex, which show selectivity for 3-containing nAChRs, provides further evidence for the involvement of 3-containing nAChRs in hippocampal [³H]DA release. Although not potent, d-TC inhibited hippocampal DA release with the same range of potency as reported for human recombinant 3-containing nAChRs (Chavez-Noriega et al., 1997; Stauderman et al., 1998). Furthermore, as observed in the present study, DHE was reported to be less potent than d-TC at human recombinant 34 nAChRs (Chavez-Noriega et al., 1997). Finally, the lack of effect with -CtxMII eliminates a role for 323 and/or 623^*.

In summary, results obtained from rank order studies support a role for 34^* nAChRs in hippocampal [³H]DA release. However, differences in pharmacology suggest that there are some differences in nAChR subtypes mediating release of each neurotransmitter. Although (±)-UB-165 elicited a full response relative to nicotine in [³H]NE release, it behaved as a partial agonist in [³H]DA release. Because the pharmacologic profile for (±)-UB-165 has not been fully characterized in rodent brain, it currently is difficult to determine other nAChRs involved in rat hippocampal [³H]DA release. Furthermore, the magnitude of blockade produced by MG 624 for each neurotransmitter suggests differences and supports the involvement of more than one receptor in hippocampal [³H]DA release. Because our studies do not reveal the likely involvement of other well established nAChR subtypes, the possibility exists that more complex heteromers, such as those containing 5 and/or 3, also may contribute to the hippocampal [³H]DA release we observe. The exact localization of nAChRs involved in DA release in the hippocampus presently is not clearly defined. DA release may be mediated directly by receptors localized on terminals of dopaminergic afferents projecting from SN and/or VTA, predominantly to the ventral hippocampus, or it could be evoked indirectly by nondopaminergic neurons that then enhance DA release from other neurons located in the hippocampus.

Interestingly, although DA functions in the regulation of different aspects of cognitive brain functions (Nieoullon, 2002) and 42 and 7 are the nAChRs identified thus far to be involved in working memory function in the hippocampus, these receptors do not seem to play a role in either hippocampal [³H]DA or [³H]NE release (Clarke and Reuben, 1996; Wonnacott, 1997; Luo et al., 1998; Anderson et al., 2000). In support of the novel findings of a significant role for 34^* nAChRs in DA release from hippocampus and the possible relevance to cognition that we describe, SIB1553A, a 4-preferring nAChR agonist (Rao et al., 2003), recently has been reported to exhibit cognitive-enhancing properties, particularly in working memory, in several models (Bontempi et al., 2003). As more selective nAChR ligands are developed, it will be worth further investigating the involvement of 34^* in cognition as well as in other brain functions that may respond to increased DA release in hippocampus.

	Acknowledgements

 
We thank Dr. Linda Werling for valuable comments and suggestions.

	Footnotes

 
doi:10.1124/jpet.104.076794.

ABBREVIATIONS: nAChR, nicotinic cholinergic receptor; DA, dopamine; NE, norepinephrine; VTA, ventral tegmental area; SN, substantia nigra; ABT-594, (R)-5-(2-azetidinylmethoxy)-2-chloropyridine; MG 624, N,N,N-triethyl-2-[4-(2 phenylethenyl)phenoxy]-ethanaminium iodine; (±)-UB-165, (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non2-ene; DMPP, 1,1-dimethyl-4-phenyl-piperazinium iodide; Hex, hexamethonium; d-TC, d-tubocurare; Mec, mecamylamine; DHE, dihydro--erythroidine; MLA, methyllycaconitine; EB, (±)-epibatidine; -CtxMII, -conotoxin MII; -CtxImI, -conotoxin ImI; -BTX, -bungarotoxin; SIB1553A, (±)-4-[2-(1-methyl-2-pyrrolidinyl)ethyl]thio]phenol hydrochloride.

Address correspondence to: Dr. Pamela Puttfarcken, Neurological Diseases Research, Global Pharmaceutical Research and Development, 100 Abbott Park Road, Building AP9A, Room 324, Abbott Park, IL 60064-6125. E-mail: pamela.puttfarcken{at}abbott.com

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