Brain Research 990 (2003) 203 208 www.elsevier.

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Pharmacological characterization of dopamine, norepinephrine and serotonin release in the rat prefrontal cortex by neuronal nicotinic acetylcholine receptor agonists
Tadimeti S. Rao *, Lucia D. Correa, Pamala Adams, Emily M. Santori, Aida I. Sacaan1
Merck Research Laboratories, 3535 General Atomics Court, San Diego, CA 92121, USA Accepted 4 August 2003

Abstract Neuronal nicotinic acetylcholine receptors (nAChRs) modulate synaptic transmission by regulating neurotransmitter release, an action that involves multiple nAChRs. The effects of four nAChR agonists, nicotine (NIC), 1,1-dimethyl-4-phenylpiperzinium iodide (DMPP), cytisine (CYT) and epibatidine (EPI) were investigated on [3H]-norepinephrine (NE), [3H]-dopamine (DA) and [3H]-serotonin (5-HT) release from rat prefrontal cortical (PFC) slices. All four agonists evoked [3H]-DA release to a similar magnitude but with a differing rank order of potency of EPIHDMPP c NIC c CYT. Similarly, all four agonists also increased [3H]-NE release, but with a differing rank order of potency of EPIHCYT c DMPP>NIC. NIC-induced [3H]-NE and [3H]-DA release responses were both calcium-dependent and attenuated by the sodium channel antagonist, tetrodotoxin (TTX) and by the nAChR antagonists mecamylamine (MEC) and dihydro-h-erythroidine (DHhE), but not by D-tubocurare (D-TC). The modulation of [3H]-5-HT release by nAChR agonists was distinct from that seen for catecholamines. DMPP produced robust increases with minimal release observed with other agonists. DMPP-induced [3H]-5-HT release was neither sensitive to known nAChR antagonists nor dependent on external calcium. The differences between nicotinic agonist induced catecholamine and serotonin release suggest involvement of distinct nAChRs. D 2003 Elsevier B.V. All rights reserved.
Theme: D-Neurotransmitters, modulators, transporters and receptors Topic: Acetylcholine receptors, nicotinic Keywords: Nicotine; Neuronal nicotinic acetylcholine receptor; Prefrontal cortex; Catecholamine and serotonin release

1. Introduction Neuronal nicotinic acetylcholine receptors (nAChRs) regulate catecholaminergic and cholinergic neurotransmission in several brain regions ([4,7,11,13,17 19,20,23,27 29], reviewed in Ref. [37]). The nAChR regulation of dopamine (DA) release has been extensively studied in the projection areas of the nigrostriatal pathway (e.g., striatum [4,7,11,13,23,28]) and/or in the mesolimbic pathway (e.g., nucleus accumbens [27]) as these pathways contain nAChRs [3]. The nAChR regulation of NE release was also examined in the hippocampus [4,17,28,35]. A
* Corresponding author. Kalypsys, Inc., 11099 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel.: +1-858-754-3300. E-mail address: trao@kalypsys.com (T.S. Rao). 1 Arizeke Pharmaceuticals Inc., 6828 Nancy Ridge Dr. Suite 400, San Diego, CA 92121, USA. 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0006-8993(03)03532-7

limited number of studies have evaluated nAChR regulation of 5-HT release in the striatum and hippocampus [10,12,15,35,36]. Several lines of evidence suggest a differential regulation by nAChRs of DA in the striatum and NE release in the hippocampus [4,28 30]. In addition, the pharmacology of nAChR regulation of hippocampal 5-HT appears to be quite different from that of NE release [15]. These results suggest that multiple subtypes of nAChRs are involved in regulating neurotransmission in different brain regions and are consistent with the molecular diversity of nAChRs [26,31,39]. To date, investigation of nAChR regulation of multiple neurotransmitters in a single brain region has not been reported and this was the focus of our investigation. The role of nAChRs in prefrontal cortical (PFC) function is of considerable interest as this region receives multiple neuronal inputs susceptible to modulation by nAChRs. The extensive cholinergic projections play an important role in

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higher brain functions, such as cognition [1,6]. Ligand binding and functional studies demonstrate nAChR localization in the PFC [16,17,34] and modulation of excitatory synaptic transmission [14,34]. Studies in which nAChR antagonists are directly injected into the rat PFC indicate a critical role of nAChR neurotransmission in information processing [8]. In addition to cholinergic projections, the PFC also receives DA projections from the ventral tegmental area, NE projections from the locus coeruleus and 5-HT projections from the raphe nucleus (dorsal and median nuclei) [5,33]. These monoaminergic systems appear to play an important role in modulation of memory fields [37]. ACh release in the PFC and subsequent activation of nAChRs can therefore differentially influence the release of a number of neurotransmitters. These activities may be of importance in understanding the cognitive enhancing activities of nicotine (NIC) and other nAChR agonists in rodents and humans [1,22]. The results have appeared in abstract form [25,30].

2. Materials and methods 1-[7,8-3H] Norepinephrine ([3H]-NE, 40 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). 3,4[7-3H] dihydroxyphenylethylamine ([[3H]-DA, 20 Ci/mmol) and, [3H]-5-hydroxytryptamine (28 Ci/mmol; 5-HT or Serotonin) were purchased from NEN (Boston, MA). ( )NIC hydrogen tartrate, mecamylamine (MEC) HCl, D-TC, 1,1-dimethyl-4-phenylpiperzinium iodide (DMPP), desipramine HCl, cytisine (CYT), tetrodotoxin (TTX) HCl were purchased from Sigma (St. Louis, MO). Dihydro-h-erythroidine (DHhE) HCl and ( F ) epibatidine (EPI) 2 HCl were purchased from Research Biochemical (RBI, Natick, MA). All other reagents were of the highest purity commercially available. 2.1. Animals Male Sprague Dawley rats (250 300 g) purchased from Harlan (San Diego, CA) were used throughout the study. The rats were acclimated to the vivarium (temperature: 22 24 jC, humidity: 50 55%, with 12-h light dark cycle) for 3 5 days before use in experiments. All the experiments were conducted as per institutionally approved animal care guidelines. 2.2. Neurotransmitter release assays Superfusion release assays in the various brain areas were conducted as previously described [28,29]. Briefly, rats were decapitated and the brain rapidly dissected on ice. The PFC slices were cross chopped (300 Am) in a McIlwain tissue chopper and equilibrated in Krebs buffer (in mM: sodium chloride, 119.5; potassium chloride, 3.3; calcium chloride, 1.3; potassium dihydrogen phosphate, 1.2; mag-

nesium sulfate, 1.2; EDTA, 0.03 and glucose 11.0; pargyline, 0.01) that was continuously gassed with 95 5% O2/ CO2 mixture for 10 min. The PFC slices were loaded with one of the following tritiated neurotransmitters (60 nM [3H]DA, 50 nM [3H]-NE and 57 nM of ([3H]-5-HT) for 30 min at 37 jC in Krebs buffer. For the [3H]-DA release assay, PFC slices were pre-incubated for 5 min at 37 jC in Krebs buffer containing desipramine (1 AM) prior to the addition of [3H]-DA. Inclusion of 5-HT uptake inhibitor during this labeling procedure did not affect the uptake of [3H]-DA to any significant extent. Therefore, all subsequent experiments for [3H]-DA release were conducted only in the presence of desipramine. Similar experiments suggested limited influence of specific DA uptake inhibitors or 5-HT inhibitors on [3H]-NE uptake or DA/NE uptake inhibitors on [3H]-5HT uptake, respectively. Therefore, PFC slices were loaded with [3H]-NE or [3H]-5HT in the absence of any uptake inhibitors. At the end of the loading period, tissue was rinsed with fresh warm Krebs buffer, transferred to chambers and continuously superfused with oxygenated buffer for 60 min. Following collection of superfusates to establish basal release, slices were exposed to test compounds for 3-min intervals. For antagonist sensitivity experiments, either MEC, DHhE or D-TC was added 3

Fig. 1. Concentration-related effects of NIC, EPI, DMPP and CYT on [3H]DA release (Panel A) or [3H]-NE release (Panel B) from rat PFC slices. Data are normalized to NIC (30 AM) and represent means F S.E.M. (n = 3 5 experiments each with two to three replicates).

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min prior to the agonist stimulation and included during the agonist stimulation. The fractional efflux of tritium was estimated as the amount of radioactivity in the superfusate fraction relative to the total amount in the tissue, multiplied by 100.

3. Results The nAChR agonists tested increased [3H]-DA and [3H]NE release from rat PFC slices in a concentration-dependent manner (Fig. 1).

In the [3H]-DA release assay, EPI was the most potent nAChR agonist among the four agonists examined ( F [3,15] = 12.4, p < 0.001; Fig. 1, panel A). All four agonists evoked [3H]-DA release to a similar magnitude. Similarly, all four nAChR agonists also increased [3H]-NE release from PFC slices in a concentration-related manner with a similar magnitude of increase (Fig. 1, panel B). EPI was the most potent agonist among the four agonists examined while NIC was the least potent agonist ( F [3,8] = 25.3, p < 0.001). NIC-induced [3H]-DA or [3H]-NE release was largely calcium-dependent (Fig. 2, panels A and D). NIC-evoked

Fig. 2. Pharmacological characterization of NIC-induced [3H]-DA or [3H]-NE release from rat PFC slices. Data represent means F S.E.M. (n = 3 4 experiments with two to three replicates). (A or E) Effect of presence or absence of 2.4 mM calcium in the superfusion buffer (*p < 0.05 vs. calcium present). (B or D) Effect of different nAChR antagonists (*p < 0.05 vs. NIC alone). (C or F) Effect of TTX (*p < 0.05 vs. NIC alone).

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Fig. 3. Concentration-related effects of NIC, EPI, DMPP and CYT on [3H]5-HT release from rat PFC slices. Data are normalized to DMPP (30 AM) and represent means F S.E.M. (n = 3 experiments with two to three replicates in each experiment).

catecholamine release was sensitive to the nAChR antagonists, MEC and DHhE, but not to D-TC (Fig. 2, panels B and E). In addition, NIC-evoked catecholamine release was sensitive the sodium channel blocker, TTX (1 AM; Fig. 2, panels C and F). In contrast to their effects on [3H]-NE and [3H]-DA release in PFC, the nAChR agonists showed a differential profile on [3H]-5-HT release (Fig. 3). DMPP elicited robust increases in [3H]-5-HT release. In contrast, EPI did not evoke any noticeable increase in [3H]-5-HT release at concentrations as high as 100 AM. DMPP-evoked [3H]5HT release was largely insensitive to the nAChR antagonists, MEC, DHhE or D-TC or to the removal of external calcium or TTX (data not shown).

4. Discussion The nAChRs are proposed to play an important role in modulating synaptic transmission by their effects on neurotransmitter release [38]. The nAChRs have a potentially enormous structural and functional diversity due to their pentameric structure with multiple genes encoding the alpha (a2 a9) and beta (h2 h4) subunits involved in the formation of heteromeric or homooligomeric receptors [26,31,39]. Neurotransmitter release studies in rodent brain slices or synaptosomes not only provide a useful means of defining function of native nAChRs, but also provide some insight into the composition of the receptors. If release is regulated by different receptors, this should reflect in differential agonist rank order of potency and efficacy, as well as differential antagonist sensitivity. It appears that different nAChRs regulate the release of a given neurotransmitter in different brain regions [4,23,38]. The results from this study demonstrate differential regulation of catecholamine and 5-HT release in the rat PFC by nAChR agonists. EPI was the most potent in evoking catecholamine release, yet it was least effective at evoking [3H]-5HT

release. The potency of EPI on PFC catecholamine release is consistent with reports that is the most potent nAChR agonist in the release of catecholamines from striatal or hippocampal preparations [23,28 30,38] as well as in in vivo assays [24]. The nAChR agonists did not show marked potency and efficacy differences in eliciting catecholamine release. Puttafarken et al. [23] reported the rank order of potency of EPI>CYT < NIC>DMPP in evoking [3H]-DA release from PFC. These differences may be related to methodological differences such as the inclusion of NE uptake inhibitor during the loading of PFC slices with [3H]-DA in the present investigation. Since both a and h subunits have been shown to contribute to agonist pharmacology [16], it is conceivable that subtle differences in the heteromeric assembly of nAChR subunits, and relative distribution of these receptors may contribute to differences in agonist rank order of potency and/or antagonist sensitivity. In situ hybridization studies indicated that DA and NE cell bodies have distinct nAChR subunit mRNA distribution (reviewed in Refs. [25,39]), and therefore, the respective projections are also likely to possess distinct nAChRs. This reflected in regional differences in nAChR-evoked catecholamine release [4,28,38]. The nAChR pharmacology of 3H]-DA and [3H]-NE release in PFC slices provides some evidence as to which nAChR subunits may be involved. Thus, at h2-containing heteromeric nAChRs, CYT functions as a partial agonist. The competitive antagonist, DHhE, is relatively more potent at inhibiting heteromeric h2-containing nAChR responses relative to those from h4-containing nAChRs [2,9]. In slice superfusion assays, DHhE significantly attenuates nAChRmediated striatal DA release without significant effects on nAChR-mediated hippocampal NE release [4,28 30]. On the other hand, another nAChR antagonist, D-tubocurare (DTC) is more effective at attenuating NIC-induced hippocampal NE release than NIC-induced striatal DA release. These data suggest that D-TC may have an opposite nAChR subunit selectivity to that of DHhE. The use of highly subtype selective antagonists such as alpha-conotoxin AuIB and alpha-conotoxin MII further support the notion that striatal DA release and hippocampal NE are differentially regulated by nAChRs [11,13,17]. The pharmacological sensitivity of nicotine-induced [3H]-DA and [3H]-NE release in PFC to DHhE, but not to D-TC, suggests the likely involvement of h2-containing nAChRs. However, the full agonist activity of cytisine in [3H]-DA and [3H]-NE release from the rat PFC slices implies that the h2 subunit combinations in the PFC are different from those in the striatum. Similarly, the lack of D-TC sensitivity of [3H]-DA and [3H]NE in PFC suggest that nAChR combinations in the PFC are also different from those involved in the hippocampal NE release. The pharmacology of nAChR agonist-induced [3H]-5HT release in the PFC is distinctly different from [3H]-NE or [3H]-DA release in the same region. Surprisingly, EPI

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was the least effective in [3H]-5-HT release with DMPP being the most efficacious and most potent of the four ligands examined. This profile does not match pharmacology of any known recombinant nAChRs. In addition, DMPP-induced [3H]-5-HT release appeared to have atypical nAChR pharmacology in that the release was insensitive to putative nAChR antagonists and was independent on extracellular calcium. In this respect, nAChR agonist-induced [3H]-5-HT release from PFC shows considerable similarities to that seen the hippocampal slices. Thus, Lendvai et al. [15] observed a lack of effect of EPI, NIC or CYT on electrically evoked hippocampal [3H]-5-HT release while DMPP evoked a robust release. DMPPevoked [3H]-5-HT release in the hippocampus was also calcium-insensitive and showed only modest attenuation by MEC at concentrations as high as 10 and 20 AM [15]. Kenny et al. [12] also reported a complex influence of nAChRs on hippocampal 5-HT release. Although considerable evidence exists for presynaptic localization of nAChRs on the cortical catecholamine terminals [3], definitive evidence for the localization of nAChRs on serotonergic terminals exists only for the striatum [21] and hypothalamus [32]. Thus, the destruction of 5-HT neurons markedly decreased [3H]-ACh binding to nAChRs in the striatum and hypothalamus, without significant reductions in the thalamus or cortex [32]. [3H]-5-HT release in this context argues that mechanisms other than receptor-mediated exocytosis are operative. In summary, the present investigation demonstrates a differential regulation of catecholamine and 5-HT release in rat PFC by nAChRs. Pharmacological manipulations that either directly activate nAChRs in PFC or indirectly activate through increases in ACh are likely to influence DA and NE neurotransmission and, to a lesser extent, 5-HT neurotransmission in the PFC. These activities, combined with increased glutaminergic neurotransmission observed with nAChR activation [34] strongly suggest that nAChRs in the PFC subserve the various components of excitatory functions such as focus/attention, abstract thinking and decision processes [34,37]. The results from this investigation implies that nicotinic agonists can differentially affect neurotransmitter release in a given brain region and that the magnitude of such responses will largely be determined by the subtype selectivity of the agonist. Acknowledgements

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