www.elsevier.com/locate/brainres

Brain Research 990 (2003) 203–208

Research report

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 EPIHDMPPcNICcCYT. Similarly, all four agonists also increased [3H]-NE release, but with a differing rank order of

potency of EPIHCYTcDMPP>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 limited number of studies have evaluated nAChR regulation

Neuronal nicotinic acetylcholine receptors (nAChRs)

regulate catecholaminergic and cholinergic neurotransmis-

sion 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 path-

way (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

0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0006-8993(03)03532-7

* 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.

of 5-HT release in the striatum and hippocampus

[10,12,15,35,36]. Several lines of evidence suggest a dif-

ferential 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 reg-

ulation 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

T.S. Rao et al. / Brain Research 990 (2003) 203–208204

higher brain functions, such as cognition [1,6]. Ligand

binding and functional studies demonstrate nAChR locali-

zation 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 tegmen-

tal 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 nic-

otine (NIC) and other nAChR agonists in rodents and

humans [1,22].

The results have appeared in abstract form [25,30].

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 meansF S.E.M. (n= 3–

5 experiments each with two to three replicates).

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 Se-

rotonin) were purchased from NEN (Boston, MA). (� )-

NIC hydrogen tartrate, mecamylamine (MEC) HCl, D-TC,

1,1-dimethyl-4-phenylpiperzinium iodide (DMPP), desipra-

mine HCl, cytisine (CYT), tetrodotoxin (TTX) HCl were

purchased from Sigma (St. Louis, MO). Dihydro-h-eryth-roidine (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 Kreb’s 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; pargy-

line, 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 Kreb’s buffer. For the [3H]-DA release assay,

PFC slices were pre-incubated for 5 min at 37 jC in Kreb’s

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 experi-

ments 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 Kreb’s 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 com-

pounds for 3-min intervals. For antagonist sensitivity

experiments, either MEC, DHhE or D-TC was added 3

T.S. Rao et al. / Brain Research 990 (2003) 203–208 205

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).

Fig. 2. Pharmacological characterization of NIC-induced [3H]-DA or [3H]-NE

experiments with two to three replicates). (A or E) Effect of presence or absence of

or D) Effect of different nAChR antagonists (*p< 0.05 vs. NIC alone). (C or F)

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 exam-

ined 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

release from rat PFC slices. Data represent meansF S.E.M. (n= 3–4

2.4 mM calcium in the superfusion buffer (*p< 0.05 vs. calcium present). (B

Effect of TTX (*p< 0.05 vs. NIC alone).

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 meansF S.E.M. (n= 3 experiments with two to three

replicates in each experiment).

T.S. Rao et al. / Brain Research 990 (2003) 203–208206

catecholamine release was sensitive to the nAChR antago-

nists, 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 antago-

nists, 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 neuro-

transmitter 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 neurotrans-

mitter in different brain regions [4,23,38]. The results from

this study demonstrate differential regulation of catechol-

amine 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 recep-

tors 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 re-

gional 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-containingheteromeric 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 nAChR-

mediated striatal DA release without significant effects on

nAChR-mediated hippocampal NE release [4,28–30]. On

the other hand, another nAChR antagonist, D-tubocurare (D-

TC) is more effective at attenuating NIC-induced hippo-

campal 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 re-

lease 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 combi-

nations 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]-5-

HT release in the PFC is distinctly different from [3H]-NE

or [3H]-DA release in the same region. Surprisingly, EPI

T.S. Rao et al. / Brain Research 990 (2003) 203–208 207

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 pharmacol-

ogy of any known recombinant nAChRs. In addition,

DMPP-induced [3H]-5-HT release appeared to have atypi-

cal nAChR pharmacology in that the release was insensitive

to putative nAChR antagonists and was independent on

extracellular calcium. In this respect, nAChR agonist-in-

duced [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. DMPP-

evoked [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 consid-

erable evidence exists for presynaptic localization of

nAChRs on the cortical catecholamine terminals [3], defin-

itive evidence for the localization of nAChRs on seroto-

nergic 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-medi-

ated 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 neurotrans-

mission in the PFC. These activities, combined with in-

creased 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 investiga-

tion 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

The authors acknowledge support and critical input from

Dr. G. Kenneth Lloyd during the course of the investigation.

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