Neuronal nicotinic receptors: from structure to pathology
C. Gotti, F. Clementi*
CNR, Institute of Neuroscience, Cellular and Molecular Pharmacology Section, Department of Medical Pharmacology
and Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy
Received 22 July 2004; accepted 29 September 2004
Available online 27 October 2004
Neuronal nicotinic receptors (NAChRs) form a heterogeneous family of ion channels that are differently expressed in many regions of the
central nervous system (CNS) and peripheral nervous system. These different receptor subtypes, which have characteristic pharmacological
and biophysical properties, have a pentameric structure consisting of the homomeric or heteromeric combination of 12 different subunits
By responding to the endogenous neurotransmitter acetylcholine, NAChRs contribute to a wide range of brain activities and influence a
number of physiological functions. Furthermore, it is becoming evident that the perturbation of cholinergic nicotinic neurotransmission can
lead to various diseases involving nAChR dysfunction during development, adulthood and ageing. In recent years, it has been discovered that
NAChRs are present in a number of non-neuronal cells where they play a significant functional role and are the pathogenetic targets in several
diseases. NAChRs are also the target of natural ligands and toxins including nicotine (Nic), the most widespread drug of abuse.
This review will attempt to survey the major achievements reached in the study of the structure and function of NAChRs by examining
their regional and cellular localisation and the molecular basis of their functional diversity mainly in pharmacological and biochemical terms.
The recent availability of micewith thegenetic ablation of single or double nicotinic subunits or point mutations have shed light on the role of
nAChRs in major physiological functions, and we will here discuss recent data relating to their behavioural phenotypes. Finally, the role of
NAChRs in disease will be considered in some details.
# 2004 Elsevier Ltd. All rights reserved.
1.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.Molecular structure of NAChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.1.Two classes of NAChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.2.The ligand binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.3.NAChR transition states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
aBgtx-nAChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.Homomeric or heteromeric receptors?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2.Calcium permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Progress in Neurobiology 74 (2004) 363–396
Abbreviations: Abs, polyclonal antibodies; b-AP, b-amyloid protein; aBgtx, aBungarotoxin; aBgtx-nAChRs, aBgtx-sensitive neuronal nicotinic
receptors; 6OHDA, 6 hydroxydopamine; ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; ADHD, attention deficit hyperactivity
disorder; ADNFLE, autosomal dominant frontal lobe epilepsy; CNS, central nervous system; aCntxMII, aconotoxin MII; DA, dopamine; dLGL, dorso lateral
geniculate nucleus; Epi, epibatidine; Kin,knockin; Ko, knock out; LPS, endotoxinpolysaccaride; MLA, methyllycaconitine; NA, noradrenaline; nAChRs, non
aBgtx-sensitive neuronal nicotinic receptors; NAChRs, neuronal nicotinic receptors; Nic, nicotine; PD, Parkinson’s disease; PET, positron emission
tomography; SCG, superior cervical ganglion; SCLC, small-cell lung carcinoma; SHR, spontaneously hypertensive rats; VOCCS, voltage operated calcium
channels; VTA, ventral tegmental area; WT, wildtype
* Corresponding author. Tel.: +39 02 50316962; fax: +39 02 7490574.
E-mail address: firstname.lastname@example.org (F. Clementi).
0301-0082/$ – see front matter # 2004 Elsevier Ltd. All rights reserved.
1.3.nAChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.1.Receptor stoichiometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.2. The role of a5 and b3 subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.3.nAChR and calcium homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.NAChR localisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.Brain cholinergic system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.Methods of study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.Brain localisation of NAChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1.Rodent distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2.nAChRs in retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.3. nAChRs in striatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.4. Primate distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.Pre- or postsynaptic distribution? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.Specificity of primate nAChR distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.Changes during development and aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.nAChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
aBgtx-nAChRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Relevance of NACHRs during ageing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.Non-neuronal localisations of NAChRs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.Lymphoid tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.Lung cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6. Vascular tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.Astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.Studies of knock out and knock in mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.Phenotype of a3, a5, b4 Ko and b2–b4 double Ko mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.Phenotype of a4 Ko and Kin mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.Phenotype of b2 Ko mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.1.Learning, memory and neuroprotection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.2.Drug reinforcement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.3.Development of the nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.4.Organisation of sleep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.Phenotype of a6 and b3 Ko mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.Phenotype of a7 Ko mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6.Phenotype of a9 Ko mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.7. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.NAChRs in pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.Diseases affecting the nervous system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.1.Age dependent disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.2.Age-independent disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.3.Age-related degenerative diseases of the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.Pathologies in non-neuronal tissues and cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.1.Lung cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.Vascular smooth muscle and endothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.Dysautonomia and blood pressure control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5.Intestinal epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .387
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .387
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396364
The cholinergic system is one of the most important and
filogenetically oldest nervous pathways. Acetylcholine
(ACh) is the neurotransmitter that is synthesised, stored
and released by cholinergic neurons, and the key molecules
that transduce the ACh message are the cholinergic
muscarinic and neuronal nicotinic acetylcholine receptors
(NAChRs). NAChRs are widely expressed in the nervous
system, where they transduce cholinergic transmission at
the synapses in the peripheral ganglia and in various brain
areas. In the central nervous system (CNS), the cholinergic
innervation acting via NAChRs regulates processes such as
transmitter release, cell excitability and neuronal integra-
tion, which are crucial for network operations and
influence physiological functions such us arousal, sleep,
fatigue, anxiety, the central processing of pain, food intake
and a number of cognitive functions (Changeux and
Edelstein, 2001; Gotti et al., 1997a; Hogg et al., 2003;
Lindstrom, 1997; McGehee and Role, 1995; Role and
Berg, 1996). Furthermore, it is becoming evident that the
perturbation of cholinergic nicotinic neurotransmission
can lead to various diseases involving NAChR dysfunction
during development, adulthood and aging (Changeux and
Edelstein, 2001; Gotti et al., 1997a; Hogg et al., 2003;
This review will attempt to survey the major achieve-
ments reached in the study of the structure and function of
NAChRs by examining their regional localisation and the
molecular basis of their functional diversity mainly in
pharmacological and biochemical terms. The recent avail-
ability of mice with the genetic ablation of single or double
nicotinic subunits (knock out, Ko) or a single gene mutation
(knock in, Kin), have shed light on the role of NAChRs in
major physiological functions and we will here discuss
recent data relating to their phenotypes. We will draw the
attention of the reader on the relatively new discovery of
NAChRs in non-neuronal cells and we will discuss their
relevance in physiology and pathology of the tissues where
they are present. Finally, the role of nAChRs in pathology
will also be considered. For the specific aspects of nAChR
physiology, cell biology, pharmacology and pathology that
are not covered by this review, we would like to draw the
Holladay, 2000; Changeux and Edelstein, 2001; Corringer
et al., 2000; Dajas-Bailador and Wonnacott, 2004; Decker
et al., 2004; Dwoskin and Crooks, 2001; Hogg et al., 2003;
Lester et al., 2003; Picciotto, 2003; Picciotto et al., 2001).
1.1. Molecular structure of NAChRs
NAChRs are a family of cationic channels consisting of
different subtypes, each of which has a specific pharmacol-
ogy, physiology and anatomical distribution in brain and
ganglia. They belong to the gene superfamily of ligand-
gated ion channels (of which muscle AChRs are the
prototype), which also includes gamma aminobutyric acid
(GABAAand GABAC), glycine and 5-hydroxytryptamine
(5-HT3) receptors (reviewed in Changeux and Edelstein,
1998; Karlin, 2002; Le Novere and Changeux, 1995).
1.1.1. Two classes of NAChRs
Earlier studies designed to characterise NAChRs were
based on binding assays with nicotinic radioligands in
different brain areas reviewed in (Lukas and Bencherif,
1992). These demonstrated that at least two distinct classes
of putative NAChRs exist in the nervous system: one
consisting of receptor molecules that bind3H-agonists with
nM affinity but not aBungarotoxin (aBgtx) (from now on
called nAChRs), and the other that bind the agonists with
mM affinity and aBgtx with nM affinity (from now on called
The pharmacological heterogeneity of NAChRs revealed
by these ligand studies was later confirmed and extended by
means of the molecular cloning of a family of genes
encoding various subunits. Twelvegenes coding for NAChR
subunits have so far been cloned and, like all of the other
members of the ligand-gated ion channel superfamily, they
encode for peptides that all have a relatively hydrophilic
extracellular amino terminal portion, followed by three
hydrophobic transmembrane domains (M1–M3), a large
intracellular loop, and then a fourth hydrophobic transmem-
brane domain (M4) (reviewed in Hogg et al. (2003); Sargent
(1993)). These subunits have a common ancestor, have been
highly conserved during evolution, and the same subunit has
more than 80% amino acid identity across vertebrate species
Fig. 1A (Le Novere and Changeux, 1995).
The genes that have been cloned so far are divided into
two subfamilies of nine neuronal a subunits (a2–a10) and
three b subunits (b2–b4) (Le Novere and Changeux, 1995;
Lindstrom, 2000). The a subunits have two adjacent
cysteines that are homologous to those present at positions
192 and 193 of the a1 subunit of muscle-type AChRs
whereas the b subunits (b1–b4) lack the pair of adjacent
cysteines (reviewed in Le Novere and Changeux, 1995;
Changeux and Edelstein, 1998). Both a and b subunits
contribute towards the pharmacological specificity of
NAChR subtypes (Luetje and Patrick, 1991).
On the basis of their different phylogenetic, functional
and pharmacological properties, the heterogeneous family
of NAChR subtypes have been divided into two main
classes: the aBgtx-nAChRs, which may be homomeric
(made up of a7–a9 subunit homo-pentamers) or hetero-
meric (made up of a7, a8 or a9, a10 subunit hetero-
pentamers), and the nAChRs, which contain the a2–a6 and
b2–b4 subunits, and only form heteromeric receptors that
bind agonists with high affinity (reviewed in Lindstrom,
1.1.2. The ligand binding sites
It is presumed that both homomeric and heteromeric
nAChRs have a pentameric structure with the subunits
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396365
organised around a central channel: the homo-oligomeric
receptors have five identical ACh-binding sites per receptor
molecule (one on each subunit) located at the interface
between two adjacent subunits, whereas the hetero-
oligomeric receptors have two a subunits and three b
subunits and therefore two binding sites per receptor
molecule located at the interface between the a and b
subunits see (Fig. 1B and C). The ACh binding site has a
principal and a complementary component. In heteromeric
nAChRs, the principal component is carried by the a2–a4
and a6 subunits with the complementary site carried by the
b2 or b4 subunits, whereas each subunit in the homomeric
receptors contributes to both the principal and complemen-
tary components of the binding site (Changeux and
Edelstein, 1998; Corringer et al., 2000) (see Fig. 1C).
Notwithstanding their initial classification in the a and b
subunit list, respectively, a5 and b3 subunits carry neither
the principal nor the complementary component of ACh
binding site and are therefore considered auxiliary subunits
A significant contribution to the identification of the
ligand binding site in NAChRs was made by the crystal
structure of the acetylcholine binding protein from the
fresh water snail Lymnaea stagnalis. This homopentameric
soluble protein is 210 residues long, binds ACh, is secreted
by snail glial cells into cholinergic synapses (Brejc et al.,
2001; Smit et al., 2001) and is analogous to the
extracellular ligand binding domain of the NAChRs.
Structural data of the crystallised acetylcholine binding
protein have revealed that the topology of the binding sites
is very similar to that predicted by mutations and computer
1.1.3. NAChR transition states
Functionally, the different NAChR subtypes can exist in
four distinct conformations: resting, open, and two
‘desensitised’ closed channel states (I or D) that are
refractory to activation on a timescale of milliseconds (I) or
minutes (D), but have a high affinity (pM–nM) for agonists.
The binding of ligands to the receptors at the neurotrans-
mitter binding site or in anyof the allosteric sites can modify
the equilibrium between the different conformational states
of the receptors. Moreover, the transition between the
different receptor states can also be regulated by receptor
phosphorylation, as has been shown in the case of muscle-
type receptors (reviewed in Changeux and Edelstein, 1998).
In heterologous systems, the expression of the a7–a9
subunits alone produces homomeric receptor channels
activated by ACh and blocked by nanomolar concentrations
of aBgtx with high Ca2+permeability and a rapid
desensitisation rate. The a7-containing subtypes account
for most of the high affinity aBgtx binding sites in the
central and peripheral nervous systems of different species.
The a8-containing receptors are only present in the chick
nervous system, where they not only form homomeric
1994; Keyser et al., 1993). The a9-containing receptors are
expressed extraneuronally and have an unusually mixed
nicotinic-muscarinic pharmacological profile (Elgoyhen
et al., 1994). a7-Containing receptors have been found in
many brain regions and are especially concentrated in the
hippocampus, where they can presynaptically facilitate the
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396366
Fig. 1. (A–C) Organisation and structure of NAChRs. (A) Schematic representation of the putative transmembrane topology of NACHR subunits. The model
shows the extracellular amino terminal portion, followed by three hydrophobic transmembrane domains (M1–M3), a large intracellular loop, and then a fourth
hydrophobic transmembrane domain (M4). (B) Pentameric arrangement of nAChR subunits in an assembled receptor. (C) Subunit arrangement in the
homomeric a7 and heteromeric a4b2 subtypes, and localisation of the ACh binding site.
release of transmitters such as glutamate or GABA
(Alkondon et al., 1996; MacDermott et al., 1999), or exert
a direct postsynaptic action by mediating fast synaptic
transmission (Jones et al., 1999; MacDermott et al., 1999).
The a7-containing receptors are also found in perisynaptic
locations, where they modulate other inputs to neurons and
activate a variety of downstream signalling pathways (Berg
and Conroy, 2002; Shoop et al., 1999). The a7-containing
receptors on autonomic ganglia are involved in fast synaptic
transmission despite their perisynaptic localisation (Dajas-
Bailador and Wonnacott, 2004; MacDermott et al., 1999).
The a10 subunit is similar to the a9 subunit by amino
acid sequence (Elgoyhen et al., 2001; Sgard et al., 2002), but
oocyte injections of the mRNA encoding the a10 subunit
subunits or b2–b4 subunits give no detectable currents. A
new current that is distinct from that of the homomeric a9
receptors was detected only when mRNAs coding for the a9
and a10 subunits were coinjected. This new current has
functional and pharmacological properties that are indis-
tinguishable from those of the endogeneous cholinergic
receptors present in cochlear hair cells, which have
transcripts for both the a9 and a10 subunit genes (Elgoyhen
et al., 2001; Sgard et al., 2002).
1.2.1. Homomeric or heteromeric receptors?
Recent studies have shown that the a7 subunit can also
form functional channels with the subunits of nAChRs. This
has been shown in oocytes in which a mutated form of the
(Palma et al., 1999), and a rat a7 subunit co-assembles with
the b2 subunit when expressed in heterologous systems
(Khiroug et al., 2002). Heteromeric
have less ACh affinity and a faster desensitisation rate than
L247Ta7 receptors, whereas the heteromeric a7b2 receptors
form channels with higher ACh affinity, a slower
desensitisation rate, and pharmacological properties that
are different from those of the a7 homomeric channel
(Khiroug et al., 2002; Palma et al., 1999).
No biochemical evidence of the presence of a7 hetero-
a7-containing subtypes (some of which have a slower
desensitisation rate and reversibly bind aBgtx) have been
described in rat hippocampal interneurons, the intracardiac
ganglion, the superior cervical ganglion (SCG) and chick
sympathetic neurons (Alkondon et al., 1997; Cuevas and
Berg, 1998; Cuevas et al., 2000; Yu and Role, 1998b) thus
suggesting that these tissues may contain heteromeric a7
receptors or alternatively transcribed a7 subunit.
1.2.2. Calcium permeability
Studies of native a7 receptors have confirmed that they
are as highly permeable to calcium as NMDA receptors but,
unlike the latter, do not require depolarisation of the plasma
membrane to promote calcium influx. It is likely that the
high degree of Ca2+permeability underlies most of their
functions: Ca2+influx can facilitate transmitter releasewhen
presynaptic a7 receptors are activated, depolarises post-
synaptic cells and acts as a second messenger to initiate
many cell processes, including those promoting neuronal
survival (Messi et al., 1997; Role and Berg, 1996). The
effects of the Ca2+entering through a7 receptors are limited
by a rapid receptor desensitisation, that prevents the
excitotoxicity of an excessive influx, which is mainly due
to a 247 leucine residue located in the second transmem-
brane region. The substitution of the leucine residue
responsible for desensitisation with threonine greatly
changes the functional and pharmacological properties of
the a7 subtype (L247T), leading to a receptor with higher
ACh affinity, a reduced desensitisation rate, and no ionic
current rectification (Revah et al., 1991). a7 receptor
functions are also modulated by divalent cations (including
Ca2+Zn2+, Mg2+, Pb2+, Cd2+) interacting with a site located
in the 160–174 region at the N terminal of the a7 subunit of
homomeric receptors, potentiates the ACh-induced response
(Hogg et al., 2003; McGehee and Role, 1995), and
extracellular Ca2+modulates both the activation and
deactivation of a7 receptors in cultured hippocampal
neurons (Bonfante-Cabarcas et al., 1996).
Although the functional and pharmacological properties
of the subtypes expressed in heterologous systems may be
influenced by the type of cells in which they are expressed
(Lewis et al., 1997) much of our knowledge concerning the
electrophysiological and pharmacological properties of
nAChR subtypes comes from these systems. Various
functional nAChR subtypes can be generated by injecting
neuronal mRNAs or cDNAs encoding a2–a4 or a6 subunits
in pairwise combinations with b2 or b4 subunits. These
different subtypes (i.e. a2b2, a3b2, a4b2, a6b2, a2b4,
a3b4, a4b4 and a6b4) have different biophysical and
of native nAChRs (Gotti et al., 1997a). Both the a and b
subunits determine the pharmacological and functional
properties of the expressed subtype: when expressed with
the b2 subunit the a2–a4 and a6 subunits all form channels
that vary in their average open times, single channel
conductance, agonist and antagonist sensitivity. b Subunits
appear to regulate the rate at which agonists and antagonists
bind and dissociate from the subtypes, and the pharmaco-
logical sensitivity of nAChRs (Papke, 1993).
1.3.1. Receptor stoichiometry
NAChRs are pentamers, but the stoichiometry of many
nAChRs remains to be fully elucidated. Biochemical and
electrophysiological approaches have shown that both chick
a4b2 (Anand et al., 1991; Cooper et al., 1991) and human
a3b4 subtypes (Boorman et al., 2000) have a stoichiometry
of 2a and 3b when expressed in oocytes injected with
cRNAs or cDNAs in a ratio of 1/1 (a/b). However, more
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396367
recent studies have shown that different classes offunctional
a4b2 subtypes are formed in oocytes when the rat a4/b2
subunit ratio is varied. When the ratio of a4/b2 is 1:9, the
subtypes generated are more sensitive to activation and
desensitise more slowly but, when the ratio is 1:1 or 9:1, the
more rapidly (Zwart and Vijverberg, 1998).
HEK cells stably transfected with the a4b2 subtype have
a large majority of receptors with low ACh affinity and slow
desensitisation but,whenthe cells are transiently transfected
with the b2 subunit, exposed overnight to nicotine (Nic) or
kept at a low temperature (29 8C), there is an increase in the
number of receptors that are more sensitive to activation
(Nelson et al., 2003). Metabolic labelling of these cells with
35S methionine has shown that the receptors have a
stoichiometry of (a4)3 and (b2)2, but long-term exposure
to Nic or to low temperature increases the number of
receptors with a high affinity for Nic and with a
stoichiometry of (a4)2 and (b2)3.
The results of all these studies clearly indicate that the
stoichiometry of heterologous subtypes is not only dictated
by their cDNA, but also by their relative ratio and possible
pharmacological treatments. However, it is not yet known
whether this plasticity also exists in neurons in vivo and
plays a role in mammalian brain, or whether more stringent
rules govern the assembly of the subtypes in native neurons.
1.3.2. The role of a5 and b3 subunits
A further complexity of the structure of nAChRs is
demonstrated by the subtypes containing the a5 and b3
subunits. Neither the a5 nor the b3 subunits can form
functional channels when co-expressed with another a or b
subunit, which is why they were long referred to as ‘‘orphan
subunits’’. They only form functional channels when are co-
expressed with both a and b subunits (Lindstrom, 2000).
The chick a5 subunit forms a functional a4b2a5 subtype
when co-expressed with the a4 and b2 subunits, and this
subtype (in which the a5 subunit participates directly in the
lining of the channel) has properties distinct from those of
the a4b2 subtype, with a higher Nic-gated conductance,
open probability desensitisation rate and Ca2+permeability,
and a higher half-maximal effector concentration (EC50) for
nicotinic agonists (Ramirez-Latorre et al., 1996). When
expressed with the a3 and b2 subunits, a5 increases
sensitivity to ACh, but this effect is not seen when the b2
subunit is replaced by b4 (Wang et al., 1996). Conversely,
the presence of the a5 subunit increases Ca2+permeability
and the rate of desensitisation in both a3b2 and a3b4
subtypes. In chick sympathetic neurons, the deletion of the
a5 subunit alters the sensitivity of native receptors to both
agonist and antagonists (Yu and Role, 1998a).
mutated form of the human b3 subunit (b3V273T) forms
functional channels in oocytes whose pharmacological and
biophysical properties are different from those of the a3b4
combination (Groot-Kormelink et al., 1998). These recep-
tors have a subunit stoichiometry of 2(a3), 2(b4), and 1(b3)
when the injected cRNA have a ratio of 1:1:20.
subunits (known as auxiliary subunits) do not directly
participate in the formation of the ligand binding site at the
interface of a and b subunits, and may occupy a position
comparable to that of the muscle b1 subunit in assembled
receptors. They may have a role in controlling ion
permeability and perhaps receptor localization.
1.3.3. nAChR and calcium homeostasis
NAChR expression studies have also demonstrated that
both heteromeric and homomeric receptors have two
important properties: (a) they are not only permeable to
monovalent cations but also to Ca2+; and (b) they are
functionally modulated by changes in extracellular Ca2+
regardless of any increase in intracellular Ca2+. In neurons,
NAChRs activation can play a relevant role in Ca2+
homeostasis and signalling not only because of the Ca2+
entry through different NAChR subtypes, but also because
NAChR depolarisation of the plasma membrane can activate
voltage operated calcium channels (VOCCs) and increase
intracellular Ca2+, and this may induce Ca2+mobilisation
from intracellular stores. The absolute quantity and strategic
localisation of Ca2+entry through NAChRs is likely to be
transmitter release, cell excitability, gene expression, cell
differentiation and survival (reviewed in Bonfante-Cabarcas
et al., 1996; Dajas-Bailador and Wonnacott, 2004; McGehee
and Role, 1995). The quantity of Ca2+in the different neuron
microdomains depends on the receptor subtypes and their
Ca2+permeability that varies and changes depending on the
different subunit combinations. The Ca2+:Na+permeability
combinations is in the range of 0.1:1.6, but close to 10:20 for
the homomeric a7 or a9 receptors (Fucile et al., 2003).
Moreover, a recent technique relying on the simultaneous
recording of fluorescent Ca2+signals and transmembrane
currents has given a more direct estimate of the Ca2+current
(in this case referred to as fractional current, Pf) through
NAChRs. These studies have confirmed that homomeric a7
This technique has also confirmed that the incorporation of
additional subunits in heteromeric receptors can change Ca2+
permeability, as in the case of the a5 subunit in the a3b4
subtype, whose presence greatly increases the calcium
2. NAChR localisation
2.1. Brain cholinergic system
In order to clarify the functions of NAChRs especially in
brain, it is useful to summarise the most important
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396368
cholinergic pathways in which NAChRs act as transducer
molecules. The brain cholinergic system is made up of a
series of closely connected subsystems consisting of eight
major and largely overlapping groups of cells, with the
dendrites of one cell contacting those of many others;
furthermore, gap junctions and dendrodendritic synapses are
relatively common. However, each cell innervates a discrete
area and tends to establish its own discrete connections.
Although individual cholinergic neurons receive only a
small number of innervating fibers, the fact that these come
from different areas of the brain means that each cholinergic
subsystem receives a large and complete set of sensory-
based information. It can therefore be postulated that this
pattern of extensive interconnections may lead to the co-
ordinated firing of a group of contiguous neurons, and hence
to the activation of different cholinergic subsystems (see for
a review Mesulam and Geula (1988); Mesulam et al. (1989);
The major cholinergic subsystems are:
Magnocellular basal complex: This represents the most
significant group of cholinergic neurons, which provide the
greatest cortical and hippocampal input. They were
identified by Meynert as large neuronal cells (30–50 mm)
that constitute 5–10% of all of the neurons in the sub-
Peduncolopontine-laterodorsal tegmental complex: This is
cholinergic neurons are concentrated in the peduncolopon-
tine tegmental nucleus and project to the thalamic nuclei and
midbrain dopamine (DA) neurons.
Striatum: This is densely innervated by cholinergic fibers
that originate from the intrinsic cholinergic neurons
constituting approximately 1% of all striatal neurons and
do not project beyond the borders of the striatum.
Lower brain stem: Cholinergic neurons are present in the
brainstem reticular formation and spinal intermediate grey
matter that innervate the superior colliculus, cerebellar
nuclei and cortex.
Habenula–interpeduncular system: This system consists of
cholinergic neurons mainly located in the medial habenula
that project to the interpeducular nucleus trough the
habenula–interpeduncular tract or fasciculus retroflexus.
The habenula receives inputs from the thalamus via the stria
terminalisand therefore isan important station trough which
the limbic system can influence the brainstem reticular
Autonomic nervous system: The preganglionic neurons in
both the sympathetic and parasympathetic systems are
cholinergic. The parasympathetic preganglionic cells are
located in a number of nuclei in the encephalic trunk and
segments S2–S4 of the spinal cord; they project long
neuritestothe parasympathetic ganglialocatedinornearthe
target organs. The preganglionic neurons of the sympathetic
system are located in a column in the intermediolateral grey
matter of the spinal cord extending from T1 to L3. They
project to the paravertebral sympathetic ganglia and each
fibre can innervate different ganglia.
NAChRs are present in neural and non-neural cells in
brain and other organs, thus indicating their pleiotropic role
in physiology and pathology.
2.2. Methods of study
The distribution of NAChRs in brain and othertissues has
been hampered by the difficulty of identifying them,
particularly their subtypes or subunits. The distribution of
subunit mRNA can be studied by means of in situ
hybridisation with selective subunit-specific probes. In the
case of NAChR protein distribution, the tools are less
1) Labelled nicotinic agents can be used to study NAChR
distribution in homogenates of discrete brain areas
(Fels et al., 1982) or histological brain sections (Adem
et al., 1989); this method is very sensitive, but its
specificity for NAChR subtypes is not very high
because no subtype-specific ligands are yet available:
3H-Nic and3H-Epi label all NAChRs (Gerzanich et al.,
1995; Gotti et al., 1997a);3H-Cyt labels the receptors
containing the a3, a4 and b2 or b4 subunits (Anderson
and Arneric, 1994);3H-aconotoxin (aCntx)-MII labels
a3 and a6 containing receptors (Nicke et al., 2004); and
125I-aBgtx labels a7–a9/10 receptors (reviewed in
(Gotti et al., 1997a).
2) Immunohistochemistry or immunopurification from
selected brain areas with subunit-specific antibodies
(Abs) give good results in terms of specificity and
sensitivity, but the available Abs should be used with
caution as not all of them have been carefully
characterised for receptor selectivity and many of them
work in a specific way only in immunoprecipitation
assays, Western blots or immunolocalisation. In order to
study the structure of native NAChRs, we have recently
prepared a series of polyclonal Abs for each receptor
subunit that specifically recognise all of the known
subunits in chick, rodent and man. The specificity of
these Abs has been tested qualitatively in Western blots
and quantitatively in immunoprecipitation experiments
using receptors labelled by nicotinic ligands present in
transfected cells or tissue obtained from Ko mice. These
Abs, together with radiolabelled nicotinic ligands and
tissues obtained from the different areas of the nervous
system have been used to quantify the expressed
receptors and/or immunopurify different subtypes (see
3) In vivo mapping using PET and specifically prepared
nicotinicligands istheonlynon-invasivemethod thatcan
be used in humans; however, it needs to be further
improved in terms of ligand specificity, and time and
space resolution (Paterson and Nordberg, 2000).
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396369
2.3. Brain localisation of NAChRs
Despite the limitations indicated above, it is possible to
draw a tentative map of brain NAChR distribution.
2.3.1. Rodent distribution
Nicotinic receptor distribution in rodent brain has been
well known for some years on the basis of data obtained
from binding studies using radioactive nicotinic drugs, in
situ hybridisation and immunohistochemistry. Only recently
has the availability of subunit-specific Abs allowed us to
map the receptor subunit localisation in different brain areas
see (Fig. 2). The most important findings emerging from
these studies and careful binding studies (Ferreira et al.,
2001; Lena et al., 1999; Perry et al., 2002) are that the most
diffuse receptor subtype in the brain is a4b2, which can also
contain a5 and the a3b4 receptors, with or without a5 are
present not only in autonomic ganglia but also in pineal
gland, the anteroventral nucleus of the thalamus, the
subiculum of the hippocampus, the medial habenula and
interpeduncular nucleus, spinal cord and retina. The a6
containing receptors, very often in conjunction with the b3
subunit, are present in the optic pathway, the locus coeruleus
and dopaminergic neurons of the mesostriatal pathways,
where they control dopamine release. The a7-containing
receptor is also a rather diffuse receptor subtype particularly
in the hippocampus, hypothalamus, cortex and motor
nucleus of the vagus nerve whereas the a9/a10-containing
the pituitary pars tuberalis, the olfactory epithelium and the
The detailed subcellular distribution of receptor subtypes
is not easy to discover as the majority of Abs are not suitable
for immunolocalisation studies. However, in some areas
(such as the mesostriatal pathways and retina), the
combination of immunoprecipitation, functional and degen-
eration techniques has made it possible to construct a
putative but reliable map of the localisation of receptor
subtypes in the soma and synaptic boutons of the different
neurons present in the area (Champtiaux and Changeux,
2002; Champtiaux et al., 2003; Klink et al., 2001; Salminen
et al., 2004; Zoli et al., 2002). Below, we describe in detail
two studies as an example of the possibilities offered by
combining immunochemical methods with biochemical and
2.3.2. nAChRs in retina
During embryonic development, chick retina expresses a
high level of both aBgtx-nAChRs and nAChRs labelled by
3H-Epibatidine (Epi) and their number increases respec-
tively ten- and six-fold from embryonic day 7 (E7) to
postnatal day 1 (P1). Retinal aBgtx-nAChRs mainly contain
a7 at E7, but there is a subsequent increase in the number of
a8-containing receptors, which are present in both homo-
meric and heteromeric a7–a8 subtypes on P1 (Gotti et al.,
1994, 1997b; Keyser et al., 1993).
Like other brain regions (i.e. optic lobe and forebrain–
cerebellum), the nAChRs expressed in E7 retina are those
containing the a4b2 subunits but, by E11, there is an
increase in a3-, b3- and b4-containing receptors. Affinity
purification of E11 b2- and b4-containing retinal receptors
showed that both populations contain the a3 and a4
subunits, but the a4 subunit is mainly associated with the b2
subunitand a3 with the b4 subunit; the b3 subunit is present
in a similar fraction of both types of receptors. After E14,
there is a considerable increase in the receptors containing
the a6 and a2 subunits, which reached a peak by P1 when
both the b2- and b4-containing subtypes are heteroge-
neously associated with the a2, a3, a4 and a6 subunits
(Vailati et al., 1999, 2000, 2003).
Immunopurification studies of P1 a6-containing recep-
tors show that they have a complex subunit composition
(with the a6 subunit being associated with the b4, b3, a3
and b2 subunits) and a particular pharmacology insofar as
they are specifically labelled with high affinity by aCntxMII
and methyllycaconitine (MLA). The a6-containing recep-
tors are only present in the retina but not in its target tissue
optic tectum (Balestra et al., 2000b), which expresses the
a4b2 subtype and a developmentally regulated a2a5b2
subtype, whereas forebrain and cerebellum coexpress the
a4a5b2 and a4b2 subtypes as also previously reported by
Conroy and Berg (1998).
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396370
Fig. 2. Analysis of nAChR subunit content in different nervous system
areas. Immunoprecipitation analysis of3H-Epi labelled nAChRs expressed
in rat cortex, hippocampus, striatum, retina and superior cervical ganglia.
The Triton X-100 tissue extracts and immunoprecipitation were carried out
as previously described (Zoli et al., 2002) using saturatingconcentrationsof
subunit-specific antibodies. Two Abs were used for each subunit (except
a2): one directed against a cytoplasmic peptide and the other against a
COOH peptide. The level of Ab immunoprecipitation is expressed as the
percentage of3H-Epi labelled receptors immunoprecipitated by the indi-
cated antibodies, taking the amount of receptors present in the Triton X-100
extract solution before immunoprecipitation as 100%.
Similar studies of rat retina have shown the presence of a
duringthe post-natal period,theincreaseinnAChRs ismuch
higher and they become predominant during postnatal
development and adulthood. This increase in rat retinal
nAChRs is due to selective increases in the receptors
containing the a2, a4, a6, b2 and b3 subunits.
Immunopurification experiments on P21 rat retina have
shown that it contains a relatively wide range of different
nAChR subtypes grouped into three populations: a6*, a4
and (non-a4–non-a6)*, which respectively
represent 26, 60 and 14% of the total P21 nAChRs (Moretti
et al., 2004). We do not know the localisation of these
subtypes, but in situ hybridisation experiments have shown
that nAChR subunit mRNAs have highly heterogeneous
distribution patterns throughout the retinal layers.
The largemajority of nAChRs in developing and adult rat
retina contain the b2 subunit; this prevalence of b2-
containing receptors appears to be mammal-specific as
Keyser et al. (2000) also found them in adult rabbit retina.
These studies suggest that, although the temporal pattern
of expression and principal subtypes expressed are species-
specific, vertebrate retina shows increased NAChR hetero-
geneity and complexity during development that is also
maintained in adulthood. Moreover, aCntxMII is the only
available tool capable of discriminating between the a6
nAChR subtypes expressed in both species.
2.3.3. nAChRs in striatum
In agreement with other pharmacological and functional
studies using a3, a4, a6 or b2 Ko mice (Marubio and
Changeux, 2000; Zoli et al., 1998; Champtiaux and
Changeux, 2002; Champtiaux et al., 2003; Salminen
et al., 2004; Whiteaker et al., 2002) (see below), we have
biochemically and pharmacogically identified two principal
nAChR populations in rat and mouse striatum: one contains
the a4 and b2 subunits, butnot the a6 subunits (a4b2*), and
accounts for approximately 70% of the nAChRs; the other
contains the a6 and b2 subunits, and accounts for
approximately 20% of the striatal receptors. These two
populations can only be distinguished pharmacologically by
aCntxMII and MLAwhich have a low (mM) affinity for the
a4b2*, but both low (mM) and high (nM) affinity for the
In addition to dopaminergic terminals, the striatum also
contains a number of non-dopaminergic cell structures. We
used denervation with the neurotoxin 6-hydroxydopamine
(6OHDA), which is selective for dopaminergic neurons, to
distinguish the nAChR subtypes expressed by the different
structures. On the basis of the changes in subunit content
observed in 6OHDA denervated striatum, we concluded that
a4b2nAChRs are expressedby bothdopaminergicand non-
dopaminergic cell types, whereas a6b2b3, a4a6b2b3 and
a4a5b2 nAChRs are only expressed by dopaminergic
terminals, and a2a4b2 nAChRs only by non-dopaminergic
The subtypes identified in striatum are summarised in
While the functional data obtained from oocytes show
that simple two-subunit nAChRs would be sufficient to
assure a nicotinic response to a target cell, the studies of
retina and striatum show that often native nAChRs contain
more than one type of a or b subunit and are therefore
structurally homologous to muscle AChRs (reviewed in
Changeux and Edelstein (1998); Corringer et al. (2000)).
These data showing that native subtypes can consist of up to
four different subunits demonstrate that the number of
biologically relevant receptor subtypes is larger than it was
previously thought. This fact has important functional
implications because the presence of a certain subunit can
modify the localisation and/or pharmacological and/or
functional properties of native receptors.
2.3.4. Primate distribution
Two classes of3H-Nic binding sites have so far been
described: a high affinity site with a Kd of 5.5 nM (range 2–
18 nM), and a low affinity sitewith a Kd of 80 nM. NAChRs
are present in a variety of brain structures, especially in the
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396371
Fig. 3. nAChR subtypes in the striatum. (A) Scheme of the mesostriatal
dopaminergic pathway. (B) Subunit composition of the functional nAChRs
expressed by dopaminergic nerve terminals (a6b2b3, a6a4b2b3, a4b2,
thalamic, neocortical and striatal regions. The number of
receptors in these areas varies from 80 to 4 fmol/mg of
protein (with a mean value of 27 fmol/mg), or from 7 to
0.8 fmol/g of tissue.
Tables 1–3 show the most relevant areas in which
NAChRs are located as described in the literature, taking
into account the results obtained by binding of radioactive
ligands and by immunolocalisation using subunit-selective
antibodies. The description of subunit distribution is still at
the beginning for the reasons mentioned above. In general,
the described regional distribution agrees well with the
results of in vivo PET studies (Nordberg, 2000).
Tables 2 and 3 show the distribution of subunit mRNA in
human brain (Breese et al., 1997; Graham et al., 2003;
Paterson and Nordberg, 2000). Receptor distribution is very
similar among primates (Han et al., 2000, 2003), although
there are important differences that are difficult to analyse in
detail because the number of subjects studied is limited, the
techniques used are not always comparable, and in situ
studies with immunohistochemistry or immunoprecipitation
have to be completed using well-controlled Abs.
On the basis of the results of in situ hybridisation studies,
it seems that the most important and diffused receptor
subtype in human brain is the a4b2, but the a4b4/b2
subtype is also present in important parts such as the
striatum, hippocampus habenula and cortex; unlike rodent
brain, primate brain has a substantial presence of a2b2,
which may be an important subtype in particular brain
regions (Han et al., 2000). There is also a selective but
important distribution of the subtype containing a6b3
subunits in the mesencephalic nuclei, particularly in the
a7 Receptors are present in the autonomic ganglia,
hippocampus and thalamic nuclei (reticular, geniculate),
and moderately dense in cerebellum, the pituitary and
pineal glands, and cortex. The a9/10 subtypes are mainly
expressed in the cochlea, as in other animal species.
Analysis of the tables in which selected receptor subunit
distribution is reported as protein or mRNA shows that
there is a discrepancy between the relative distribution of
protein and mRNA. It is important to recognise a
discrepancy between protein and mRNA expression as
several times distribution in subunit mRNA is taken as
subunit distribution. This discrepancy could be due to the
fact that subunits may be synthesised in the neuronal soma
in one brain area and transported to other locations in
presynaptic structures, or that the different subunits have a
different turnover and assembly rate, or that mRNA for the
different subunits have a different stability or transcription
2.4. Pre- or postsynaptic distribution?
Anatomical and functional evidence suggests that
NAChRs are preferentially located at the presynaptic
boutons regulating neurotransmitter secretion in several
parts of the brain (see Wonnacott, 1997; Wonnacott et al.,
1995). In particular, presynaptic NAChRs have been
implicated in the release of ACh (Wilkie et al., 1993),
noradrenaline (NA) (Clarke and Reuben, 1996), DA (Grady
et al., 1992; Rapier et al., 1990; Wonnacott et al., 1990),
glutamate (McGehee and Role, 1995; Alkondon etal., 1997)
and GABA (Yang et al., 1996). Very recently, a method has
been developed to detect presynaptic NAChRs in neurons in
vitro (Girod et al., 2003). In rodents and monkeys, some
attempts have been made to determine the subtypes present
in presynaptic boutons and it has been found that their
subunit composition varies in different brain areas.
In addition to controlling and modulating the release of
various neurotransmitters, presynaptic NAChRs can play
other roles under particular conditions (i.e. denervation and
development), such as path finding and neuritogenesis (Role
and Berg, 1996; Zheng et al., 1994).
There is evidence that high affinity Nic binding NAChRs
are located on postsynaptic membranes in the somatoden-
dritic regions of various brain areas (Clarke, 1993), and that
Nic can elicit a cell response through postsynaptic neuronal
receptors (de la Garza et al., 1987). The fast synaptic current
in autonomic ganglia is mediated through a3b4/a5
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396372
Distribution of nicotinic receptors in primate brain (human ‘‘+’’, monkey
+ 0 0 0
0 0 0
Putamen-caudate+++ 0++ 0 0++
CA pyramid lay
++ 0 0
0 0 0
+++ 0 0 0
+ 0 0 00 0 0
0 0 0
0 0 0
0 0 0 0
0 0 0
0 0 0
0 0 ++
Pineal gland0 0 0
Medial habenula0 0 0 0
+++ 0 0 0
0 0 0
0 0 0 0
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396373
Nicotinic receptor subunit mRNA distribution in human brain
  
[2, 4, 5, 13]
[2, 5, 6, 8, 9]
[2, 5, 8, 9, 12]
[8, 9, 11]
[2, 11, 12]
[1, 12][1, 12] [1, 12]
[8, 9, 13]
  
Midbrain[1, 12] [1, 12] [1, 12]
 [2, 11–13]
[1, 10, 11, 13]
 [1, 12]
Cerebellum[2, 6][2, 4, 6] [1, 2, 6]* 
Thistableis updatedfrom Graham,A.J.,et al.,2002.Curr.Drug.Targets CNSNeurolDisord1,387–397.Insitu hybridisation andRT-PCRstudiesare included
(tissue from prenatal to elderly individuals). Where no specific sub-region is defined, either the whole region was homogenised for RT-PCR analysis or no area
in table are as follows:
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2. Hellstrom-Lindahl, A., et al., 1999. Brain Res. Mol. Brain Res. 66, 94.
3. Wevers, A., et al., 1994. Brain Res. Mol. Brain Res. 25, 122.
4. Agulhon, C., et al., 1998. Brain Res. Mol. Brain Res. 58, 123.
5. Wevers, A., et al., 2000. Behav. Brain. Res. 113, 207.
6. Hellstrom-Lindahl, E., Court, J.A., 2000. Behav. Brain Res. 113, 159.
7. Schroder, H., et al., 2001. Brain Res. Dev. Brain Res. 132, 33.
8. Breese, C.R., et al., 2001. J. Comp. Neurol. 387, 385.
9. Agulhon, C., et al., 1999. Neurosci. Lett. 270, 145.
10. Tohgi, H., et al., 1998. Neurosci. Lett. 245, 139.
11. Guan, Z.Z., et al., 2002. Brain Res. 956, 358.
12. Hellstrom-Lindahl, E., et al., 2001. Neuroscience 105, 527.
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14. Terzano, S., et al., 1998. Brain Res. Mol. Brain Res. 63, 72.
15. Tohgi, H., et al., 1998. Brain Res. 791, 186.
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17. Guan, Z.Z., et al., 1999. Neuroreport 10, 1779.
18. Martin, Ruiz, C.M., et al., 1999. J. Neurochem. 73, 1635.
19. Sparks, D.L., et al., 1998. Neurosci. Lett. 256, 151.
20. Burghaus, L., et al., 2000. Brain Res. Mol. Brain Res. 76, 385.
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22. Banerjee, C., et al., 2000. Neurobiol. Dis. 7, 666.
23. Burghaus, L., et al., 2003. Parkinsonism Relat. Disord. 9, 243–246.
24. Mousavi, M., et al., 2003. Neuroscience 122, 515.
25. Perry, E.K., et al., 2001. Am. J. Psychiatr. 158, 1058.
26. Guan, Z.Z., et al., 2000. J. Neurochem. 74, 237.
27. Martin Ruiz, C.M., et al., 2000. Neuropharmacology 39, 2830.
28. Graham, A.J., et al., 2003. J. Chem. Neuroanat. 25, 97.
29. Martin-Ruiz, C., et al., 2002. Neurosci. Lett. 335, 134.
30. Graham, A., et al., 2002. Neuroscience 113, 493.
subtypes. In agreement with a postsynaptic localisation of
AChRs are the data of Del Signore et al. (2002, 2004)
concerning the rodent cervical superiorganglion (SCG), and
Horch and Sargent (Horch and Sargent, 1995) in chick
ciliary ganglion, who found that mAb35-AChRs are located
in both synaptic and perisynaptic sites on the surface of
ciliary ganglion neurons, and that their activity is blocked by
neuronal bungarotoxin (NBT) (Chiappinelli, 1983). Simi-
larly, using a monoclonal antibody that recognises neuronal
AChRs (WF6), Schroder et al. (1989) found that NAChRs in
the cortex are located in the postsynaptic thickening. The
location of aBgtx binding sites corresponding to the human
a7 subtype has been studied in man only at cellular level
(Ciminoetal., 1992),buttheresultsofstudiesofrat andfrog
SCG (Smolen, 1983; Marshall, 1981), and chick ciliary
ganglion (Horch and Sargent, 1995; Zhang et al., 1996)
make it possible to suggest that they are located in the
1994) they have been found at both synaptic (Marshall,
1981; Smolen, 1983) and non-synaptic sites, where they can
transduce synaptic and/or regulatory signals. The aBgtx-
AChRs on chick ciliary ganglion are mainly located in
patches around the synaptic sites (Horch and Sargent, 1995;
Zhang et al., 1996). Even in this non-canonical location,
aBgtx-AChRs are capable of generating postsynaptic
currents, thus indicating that the membrane domain
regulating membrane potential is broader than previously
thought. Moreover, it is possible to speculate that, in
addition to controlling rapid communications between pre-
cell functions by increasing Ca2+influx (for example,
The predominantly presynaptic localisation of NAChRs
on nerve terminals containing different neurotransmitters,
and the presence on the same boutons of different NAChR
subtypes, is the basis of the pleiotropic effects of nicotinic
drugs that modulate several pathways. This aspect is
therefore crucial in the development of selective nicotinic
drugs for pharmacological and therapeutical purposes.
2.5. Specificity of primate nAChR distribution
Although the distribution of receptors in primates is still
not completely known (particularly in the case of human
receptors and the distribution of receptor subtypes), the
available data suggest that, overall, it is not greatly different
from that in rodents. However, there are some discrepancies:
e.g. a2 mRNA is more widely distributed in primate than
rodent brain and the binding of nicotinic drugs correlates
well with this subunit distribution, whereas it is known that
the main nicotinic binding site in rodents is due to a4b2
receptors; b2 is the most widely expressed subunit in rat
in human brain is less ubiquitous; aBgtx binding and a7
distribution is more diffuse in monkey than in rodent brain,
thus suggesting that this receptor subtype plays an important
role in monkey brain function. It is conceivable that future
studies in this field will reveal further species-related
differences. Knowledge of receptor subtype distribution is
important in order to allow the correct correlations to be
made between receptor subtypes and brain functions or
pathologies, which would assist in creating valid animal
models of human brain pathologies and finding subtype-
specific therapeutic agents.
3. Changes during development and aging
NAChRs change considerably during development and
aging in all animal species. The earliest detection of
nAChRs, as mRNA or as ligand binding, is on E7 in chick
(Vailati et al., 2003), E11 in rat (Zoli, 2000a,b) and after 5–7
weeks of gestational age in human brain (Zoli, 2000b).
Studies of3H-Nic and125I-aBgtx binding sites and mRNA
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396 374
Nicotinic receptor subunit protein expression in human brain*
[11, 16, 18–23]
[16, 18, 20–24] [11, 19]
  [27, 29]
Cerebellum   
This table is updated from Graham, A.J., et al., 2002. Curr. Drug. Targets CNS Neurol Disord 1, 387–397. For references in table, please see legend of Table 2.
*From immunohistochemical (describing neuronal immunoreactivity) and Western blot studies.
expression for the individual subunits during development
have shown that they have different temporal and spatial
In rat, the mRNA for the a2, a3, a4, a5 and a6, and the
b2 and b4 subunits, are present from the early moments.
Their brain distribution is not dissimilar from that of adult
animals, with high intensity in the diencephalon, brainstem
and spinal cord, and lower intensity in the telencephalic
structures; however, the temporal expression of the
individual subunits may vary because, for example, b4
precedes a3 by two days, but they then colocalise. The
postnatal behaviour of the subunits is particular in the sense
that many of them (notably a3 and a4, b2) are suppressed
early in development and increase later in postnatal life
(Cimino et al., 1994; Shacka and Robinson, 1998; Zoli,
2000b; Zoli et al., 1995). This temporal behaviour depends
on the different brain structures: for example, in cortex and
the expression of a4 is already high at P7.
In humans, high-affinity
steadily during gestation (from 12 to 27 weeks), reaching
higher levels than at any other time during life (Cairns and
Wonnacott, 1988; Court and Clementi, 1995; Court et al.,
1995). The highest concentrations have been observed in the
nucleus basalis of Meynert and the tegmental nuclei,
followed by the globus pallidus, the putamen, the cerebellar-
relay nuclei, the parietal and cerebellar cortex, the thalamus
and the spinal cord; the lowest levelisin the medulla (Cairns
and Wonnacott, 1988; Court et al., 1995; Kinney et al.,
1993). During the perinatal period and early infancy, the
concentration of NAChRs in the different brain areas
decreases considerably, with the exception of the major
cerebellar nuclei in which the concentration of receptors
There is general agreement that Nic binding and the
expression of subunit mRNA slightly decrease in rat during
aging, although with some regional specificity. For example,
a4 mRNA decreases in the thalamus but not in cortex, and
the decrease in a3 is more marked than that of a4 and
present in nearly all of the brain zones assayed except for the
medial habenula (Ciminoet al., 1994). The finding that there
is marked neurodegeneration in b2 Ko mice during aging
indicates that b2- containing receptors are important for
neuronal survival during aging (Zoli, 2000b).
There is general consensus that the concentration of Nic
binding sites steadily decreases throughout life in nearly all
of the studied human brain regions. The reported
discrepancies are probably due to difficulties in comparing
different clinical populations. One exception could be the
thalamus, in which the binding sites remain constant or
increase (Court and Clementi, 1995; Flynn and Mash, 1986;
Nordberg et al., 1992). In the case of subunit distribution,
there is a general agreement that the b2 subunit decreases
3H-Nic binding increases
with age in all brain structures, but a4 and a3 do not
decrease in the hippocampus and putamen.
In rat fetal brain,125I-aBgtx binding sites and mRNA for
a7are present from E13 toE14 and increase untilbirth,after
which, they decrease in the first days to adult expression
levels. They are restricted to specific areas, such as the
hippocampus and the dorsal motor nucleus of the vagus
nerve (Tribollet et al., 2004). In human fetal brain,125I-
aBgtx binding sites are present as early as 5–7 weeks of
gestional age and subsequently increase steadily. They are
high in the pons, medulla oblongata, mesencephalon,
cerebellum and spinal cord (Falk et al., 2002).
After birth and throughout life (between 60 and 90 years
of age), their concentration slowly decreases in the
hippocampal CA1 region and the entorhinal cortex (Court
and Clementi, 1995), the thalamus (Nordberg and Winblad,
1986), and the striatum (Schulz et al., 1993) but remains
constant in the frontal cortex and cerebellum (Falk et al.,
3.3. Relevance of NACHRs during ageing
These findings indicate that high affinity Nic and aBgtx
binding sites are independently regulated among species
during the development and aging of different brain areas.
The different transcriptional regulation of NAChR subunits
is probably due to gene promoter structure and different
transcription factors, which may be different among subunit
genes and brain areas (Gotti et al., 1997a). The described
changes during development and aging are specific to
NAChRs because, for example, the changes in muscarinic
and glutamatergic receptors, as well as in choline
acetyltransferase activity, follow a different pattern (see
Court and Clementi (1995) for more details). The pattern of
nAChR expression over time suggests that these receptors
concentration is high during the stage of synapse formation.
In vitro experiments suggest that NAChRs (particularly a7)
may control the development of neuronal architecture,
stabilise synapse formation, and orient and control neurite
outgrowth (Lipton and Kater, 1989; Pugh and Berg, 1994;
Quik, 1995; Role and Berg, 1996; Zheng et al., 1994).
These dataindicate thatNAChRs areofgreatrelevancein
two critical periods for brain life: early pre- and perinatal
circuit formation, and cell degeneration during aging. The
useof nicotinic drugs inthesetwo periods couldthereforebe
very important. The exposure of fetal brain to Nic, as result
may greatly modify brain circuitry as it is known that the
administration of nicotinic drugs at this stage can also
increase the number of NAChRs (Narayanan et al., 2002)
and particularly of some receptor subtypes, and that
NAChRs are important in establishing synaptic connections
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396375
(Role and Berg, 1996). Furthermore, the use of nicotinic
drugs to prevent or decrease the brain degeneration and
extremely important and positive aim to pursue. However,
the experimental and human data are still not complete and
do not allow a rational intervention. Is it better to stimulate
all of the receptor subtypes, or only the homomeric a7
(hippocampus) or those that are more decreased (the b2-
containing receptors) or those that are expressed in
particular regions (a6b3 in dopaminergic regions)? These
therapeutic dilemmas will be easier to approach when more
information is available especially in human beings.
4. Non-neuronal localisations of NAChRs
ACh is probably the oldest signalling molecule, and
appeared very early in evolution before nervous cells. It is
surprising that it is still involved in cell-to-cell commu-
nications in various non-neuronal tissues and controls
important cell functions such as proliferation, adhesion,
migration, secretion, survival and apoptosis in an autocrinal,
justacrinal and paracrinal manner (Grando et al., 1993;
Sastry et al., 1979). Many of these functions are mediated
through NAChRs on non-neuronal cells, and it is clear that
they can play an important role.
mRNAs coding for the a4, a5, a7, b4 and b2 subunits
have been found invertebrate adult muscle (Corriveau et al.,
1995; Maelicke et al., 2000; Sala et al., 1996), in a human
rat muscle. a7 is highly expressed in mammalian muscle
during development and the perinatal period, and decreases
later on in adult life. It is upregulated by denervation
(Maelicke et al., 2000). The function of NAChRs in muscle
is not known, but it is possible that they may control various
metabolic and trophic functions, and perhaps gene expres-
sion in areas that will receive the incoming nerve fibres, by
increasing Ca2+influx. The innervating fibres contain
presynaptic a3b2 subtype receptors whose activation
facilitates ACh release (Faria et al., 2003).
4.2. Lymphoid tissue
et al., 1990), and in circulating and thymic T lymphocytes
(Paldi-Haris et al., 1990); and their number increases during
aging (Nordberg et al., 1990). Ligand binding, RT-PCR, or
Southern or Western blotting studies have shown that human
T lymphocytes, and lymphocyte cell lines such as Jurkat,
Molt4 and H9, as well as hypertrophic human thymi
(Mihovilovicetal., 1993),expressthe a3,a4,a7, b2and b4
receptor subunits (Kuo et al., 2002; Sharma and Vijayar-
aghavan, 2002); the receptor subtypes likely to be expressed
are a3b4, a4b2 and a7. The thymus receptors are located
in T cells and epithelial cells (Kuo et al., 2002); and,
although their function in human lymphoid tissue is
unknown, their location and modulatory effects on the
proliferation of thymic and circulating T lymphocytes
(Middlebrook et al., 2002; Richman and Arnason, 1979;
Rinner et al., 1994) suggest that they may affect T cell
proliferation, as well as thymic differentiation and selection
processes. Functional NAChRs containing either a4 or a7
subunits are also present on B cells, where they stimulate
growth and decrease antibody production (Skok et al.,
2003). Lymphocytes possess cholinoacetyltransferase, acet-
ylcholinesterase (AChE) and vesicular acetylcholine trans-
porter (Kawashima andFujii,2003), andso it ispossiblethat
ACh may activate nAChRs via an autocrine pathway. The
presence of NAChRs in peripheral blood cells have spurred
brain NAChRs to diseases and drug treatments (Benhammou
et al., 2000; Perl et al., 2003). The NAChRs of peripheral
blood cells respond to drugs and are modified in some
diseases, but too little is known about the mechanisms
regulating the expression of central and peripheral receptors
tobeable toproposeperipheralreceptors asareliablemarker
gene is differently regulated in neurons and lymphocytes
(Battaglioli et al., 1998).
It has long been known that circulating phagocytic cells
have NAChRs (Davies et al., 1982), but it has only recently
become evident that human macrophages have a7 NAChRs,
and that their activation by Nic reduces the release of TNFa
and interleukins 1 and 6 induced by the endotoxin
polysaccharide (LPS) (Wang et al., 2003a). It is also known
that vagal stimulation inhibits the secretion of pro-
inflammatory cytokines and reduces the inflammatory
process, and that this does not occur in animals deficient
of a7 receptors (Borovikova et al., 2000). Mice alveolar
macrophages express the a4b2, but not the a7 subtype and,
also in this case, activation by Nic down-regulates the
production ofinterleukins 6, 12 and TNFa,thus allowing the
proliferation of infecting bacteria (Matsunaga et al., 2001).
It has very recently also been shown that brain microglia
have a7 receptors and that Nic inhibits LPS-induced TNFa
release in microglial cells in vitro (Shytle et al., 2004). The
presence of a cholinergic anti-inflammatory pathway in
different tissues makes it easier to understand: (a) the
paradoxical protective effect of smoking on immune-
mediated lung diseases (e.g. immune alveolitis) and its
negativeeffect on bacterial infection that leads to its positive
(b) the anti-inflammatory activity of Nic in inflammatory
bowel disease; and (c) the neuroprotective effect of Nic in
degenerative diseases of the CNS associated with chronic
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396 376
inflammation. These findings suggest a possible new
therapeutic approach to a variety of inflammatory condi-
Human epidermal keratinocytes express nAChRs that
have the biophysical and pharmacological properties of an
a3 subtype (Grando et al., 1995). Furthermore, the presence
of a3, a7, a9, b2 and b4 subunits in these cells has been
shown by means of antibody binding, RT-PCR experiments
and Ca2+influx (Grando, 2001; Zia et al., 2000). The a3
subunit is more expressed in small cells, localised in
membranes forming cell junctions, and the a7 in large
differentiated cells (Zia et al., 2000). NAChRs mediate
several skin functions, particularly keratinocyte prolifera-
tion, apoptosis, differentiation, adhesion and motility,
mainly by regulating Ca2+influx in keratinocytes (Grando
et al., 1995; Arredondo et al., 2002; Grando, 2001; Nguyen
et al., 2003; Zia et al., 2000). As ACh is synthesised and
released by human keratinocytes (Grando et al., 1993), and
skin is also innervated by cholinergic fibers, ACh can
activate NAChRs via paracrine-autocrine pathways. All of
these nicotinic actions are important in controlling skin
physiology and development, particularly the formation of
an efficient skin barrier (Burkhart and Burkhart, 2001).
known that smoking has a positive effect on pemphigus
flaring (Wolf et al., 1998); furthermore, Nic can have
positive effects on other dermatological diseases and wound
4.5. Lung cells
ACh is synthesised, released and destroyed in various
cells throughout human airways, as well as in effector cells
effects are mediated by muscarinic and NAChR (Wessler
and Kirkpatrick, 2001). a3, a5 and a7 NAChR subunits are
present in bronchial epithelial cells, a4 in alveolar epithelial
cells (Zia et al., 1997) and a4, a7 and b2 in neuroepithelial
bodies (Fu et al., 2003). Various nicotinic receptor subunits
(a3, a5, a7, b2, b4), aBgtx binding sites and the capacity to
synthesise, release and degrade ACh (Song et al., 2003) are
also present in pulmonary neuroendocrine cells and in the
human small-cell carcinoma cell lines derived from them
(Chini et al., 1992; Cunningham et al., 1985; Maneckjee and
Minna, 1990; Quik et al., 1994; Schuller et al., 2003; Song
et al., 2003; Tarroni et al., 1992). Nicotinic stimulation in
these cells induces the secretion of neurotransmitters and
increases cell proliferation (Cattaneo et al., 1993; Klapproth
et al., 1998; Quik et al., 1994; Schuller et al., 2003; Song
et al., 2003). The presence of nAChRs in lung is important
because the Nic in cigarette smoke reaches lung cells at high
concentrations and may play a role in stimulating thegrowth
of small-cell lung carcinoma, an extremely aggressive
tumour associated with tobacco abuse (Weiss, 1991). In a
fetus exposed to maternal or passive smoking, Nic can alter
lung development and cause lung hypoplasia with a
reduction in the complexity of the gas-exchange surface
(Sekhon et al., 1999). On the other hand, nAChRs may be a
relevant pharmacological target in the case of proliferative,
immunological and developmental diseases of the lung.
4.6. Vascular tissue
Nic is a potent stimulus of angiogenesis and increases
proliferation in endothelial cells acting through NAChRs
(Heeschen et al., 2001; Villablanca, 1998). The vascular
system contains a number of nicotinic subunits in
endothelial cells (a3, a5, a7, a10, b2, b and b4) and
vascular smooth muscle (a2, a3, a4, a5, a7, a10)
(Bruggmann et al., 2003; Macklin et al., 1998; Wang
et al., 2001). Smooth muscle cells selectively express
NAChR subtypes depending on the tissue localisation of the
vessels. a3 and a5 are widely distributed among arteries but
present in muscle, kidney or lung small arteries; a7 is
widespread but lacking in the renal circulation. NAChRs are
present in arteries devoid of cholinergic innervation, but it
has been reported that endothelial cells can synthetise,
release and degrade ACh (Kawashima et al., 1990).
Although the in vivo function of arterial NAChRs is not
yet known, it is possible to postulate that they may play an
important role in controlling angiogenesis and smooth cell
proliferation, thus suggesting the obvious therapeutic
application of nicotinic drugs in arteriosclerosis, tumour
growth, and revascularisation after ischemic insults. If the
specific complement of NAChRs in individual arteries is
confirmed, we can expect to exert possible selective
interventions in particular organ vessels.
Brain endothelial cells, an important component of the
blood–brain barrier, express the a5, a7, b2 and b3 nicotinic
subunits (Abbruscato et al., 2002). It is known that Nic alters
the permeability of the blood–brain barrier, which could be
mediated by a decrease in the expression of a7 and b2
et al., 2002). These findings suggest that NAChRs are
involved in controlling this important function and should be
borne in mind when the neuronal effects of smoking are
considered, when drugs acting on the CNS are given to
smokers, and when it is convenient to modify pharmacolo-
gically the permeability of the blood–brain barrier.
many years ago (Hosli et al., 1988), but it has only recently
been confirmed and studied in more detail. a3, a4, a7, b3
and b4 subunits have been detected in hippocampal
astrocytes (Graham et al., 2003; Sharma and Vijayaragha-
van,2002), and a7seems to bethe mostimportant. Inmouse
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396377
hippocampal astrocytes and the susceptibility of mice to Nic
seizures (Gahring et al., 2004b). Some data indicate that
a4b2 receptors exist in glial processes, and a3b4 receptors
in glial soma (Graham et al., 2003). Spinal chord astrocytes
also have a complement of a3, a5 and b2 subunits (Khan
et al., 2003). The real role, relevance and functions of these
receptors in astrocyte life or astrocyte-neuron relationships
are not known, but the presence of NAChRs in astrocytes is
in line with an integrated view of synaptic activity as being
not only confined to neuron–neuron interactions, but also
consistently modulated by perisynaptic astrocytes. This
view opens the way to the exploration of new pharmaco-
logical interventions aimed at influencing synaptic activity
and modifying astrocyte function in pathologies with
5. Studies of knock out and knock in mice
The use of genetically engineered Ko or Kin in which one
or more genes of interest are silenced or mutated provides a
unique opportunity to analyse the pharmacology and
functional role of NAChRs in complex neurobiological
The use of Ko mice may have some drawbacks because
their lack of the subunit of interest may lead to some forms
of adaptation during development, with the up- or down-
regulation of some receptor subtypes that may confound the
interpretation of behavioural changes. Only future studies of
animals with the conditional or inducible Ko of individual
subunits will help us to draw final conclusions concerning
the role of individual subunits in behaviour.
The following nAChR subunits have so far been knocked
out: a3–a7, a9, b2–b4. Only the a3 subunit appears to be
necessary for survival as the mice lacking the other
subunits are all viable and appear grossly normal. We
here mainly review the recent results obtained in Ko and Kin
mice (for more exhaustive review, see (Champtiaux et al.,
2003; Cordero-Erausquin et al., 2000; Drago et al., 2003;
Lester et al., 2003; Marubio and Changeux, 2000; Picciotto
et al., 2000, 2001; Picciotto and Corrigall, 2002; Wang et al.,
5.1. Phenotype of a3, a5, b4 Ko and b2–b4 double
The a3 subunit is highly expressed in the autonomic
ganglia, but is also found in subsets of neurons in the medial
habenula, dorsal medulla and retina. a3 Ko animals usually
die in the first week of life due to multiorgan dysfunction,
with impaired growth and increased mortality before
weaning and a phenotype very similar to that of the double
b2–b4 Ko mice (Xu et al., 1999a,b). Both a3 Ko and double
b2–b4 Ko mice have an enlarged bladder and develop
bladder infection, dribbling urination and urinary stones.
In the SCG, both the b2 and b4 subunits are associated
with a3 subunits (Del Signore et al., 2002, 2004) and, in
heterologous systems, both form functional channels with
the a3 subunit (McGehee and Role, 1995). Nic-induced
whole-cell current is abolished in the SCG neurons of a3 Ko
and double b2–b4 Ko mice but, although reduced, it is still
present in the SCG neurons of b4 Ko mice (Xu et al.,
1999a,b). These results suggest that the a3 subunit may
combine with the b2 subunit in b4 Ko mice, and this may be
sufficient for functional compensation in the SCG.
Studies of b4 Ko mice have shown that the deficiency
affects ganglionic transmission and leads to an attenuated
bradycardic response to high-frequency vagal stimulation,
and significantly reduced ileal and bladder contractile
responses to nicotinic agonists (Wang et al., 2001, 2003b).
These and the SCG results together strongly support the
hypothesis that the lack of the b4 subunit impairs nicotinic
conductance in both sympathetic and parasympathetic
ganglia, and that ganglionic nAChR is formed at least by
a3 and b4 subunits.
Quantitative autoradiography studies of the brains of a3
Ko mice (Whiteaker et al., 2002) have shown that, unlike
what was thought on the basis of the results of expression
studies in heterologous systems (Cartier et al., 1996), the
large majority of the high affinity aCntxMII binding sites is
unchanged and there is only a reduction in the habenula
interpeduncular tract, thus suggesting that this is the major
site where the a3b2 subtype is expressed and contributes to
aCntxMII binding. These results, together with those
relating to a6 Ko mice indicate that the large majority of
the aCntxMII binding sites in rodent brain are a6-
Studies of a5 Ko mice have revealed impaired cardiac
parasympathetic ganglionic transmission and increased
sensitivity to hexamethonium (Wang et al., 2002a). More-
over, although they have no visible phenotype with normal
baseline behaviours, they are less sensitive to Nic-induced
seizures and behaviours related to locomotor activity than
WT mice (Salas et al., 2003). b4 Ko mice are even more
resistant to Nic-induced seizures than a5 Ko mice, and
double a5/b4 Ko mice are more resistant than either single
Ko animal (Kedmi et al., 2004). Furthermore, both a5 and
a5/b4 Ko mice showed a significantly shorter latency time
to seizures than WT mice.
5.2. Phenotype of a4 Ko and Kin mice
a4 Ko mice show dramatically reduced antinociceptive
effects of Nic in hot-plate test (primarily brain-mediated
pain response) and a slightly reduced analgesic response in
response) (Marubio et al., 1999).
The same pattern is seen in b2 Ko mice, although higher
doses of Nic have a residual analgesic effect during
both tests. Binding and electrophysiological studies of the
thalamus and raphe magnus of a4 and b2 Ko mice have
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396 378
revealed the disappearance of high affinity agonist binding
and the loss of Nic-evoked currents. On the contrary, the
sensory neurons of the dorsal horn of the spinal cord showed
a Nic-dependent increase in the current frequency, probably
due to the presence of an a3b4 subtype.
By acting through different nAChR subtypes, nicotine
can decrease locomotor activity in a novel environment and
increase locomotion via activation of dopaminergic path-
ways in a familiar environment. Marubio et al (1999, 2003)
found no difference in basal locomotor activity between a4
wildtype (WT) and Ko mice in either environment, but an
independent line of a4 Ko mice showed increased
locomotion, sniffing and total rearing, and an increase in
the basal level of anxiety (Ross et al., 2000).
behavioural models of anxiety and depression have shown
that Nic can have anxiolytic and antidepressant effects
containing receptors in the brain are associated with the b2
subunit, the fact that no difference in anxiety was found in
b2 Ko mice is surprising, and can perhaps only be explained
by differences in the genetic background of the a4 and b2
Ko mice, or by the fact that a non-a4b2 nAChR, present in
a4 Ko but absent in b2 Ko has ansiogenic effects (Picciotto
et al., 1995).
Epidemiological evidence suggests that smoking can
protect against Parkinson’s disease (PD) (Fratiglioni and
Wang, 2000), a hypothesis that has also been tested using a4
Ko mice in an animal model in which acute Nic pre-
treatment significantly inhibited methamphetamine-induced
nigrostriatal neurodegeneration in WT but not a4 Ko mice
(Ryan etal., 2001). The role of a4-containing receptor in the
neuroprotection of the dopaminergic system was also
examined using a4 9’S Kin mice (Labarca et al., 2001),
which express nAChRs with a mutation in the transmem-
brane M2 domain of the a4 subunit that makes them more
agonist sensitive. Even in the hemizygous state, the Kin
mutation led to a late embryonic loss of mid-brain
dopaminergic neurons. If the expression of this mutated
subunitisdecreasedby the inclusion ofaneomycin-resistant
cassette in an intron, the mice survive, and electrophysio-
logical recordings from mid-brain neuroprogenitor cells are
which, at low concentrations, is also an agonist of mutated
(a4 9’S)b2 receptors (Fonck et al., 2003). In brief, these
studies indicate that the activation of a4-containing
receptors can be neuroprotective, but their hyperactivation
can lead to neurodegeneration.
5.3. Phenotype of b2 Ko mice
b2 subunit was the first of the nAChRs subunit to be
knocked down. The b2 Ko mice have lost the vast majority
of high affinity nAChRs and Nic-elicited currents in neurons
from various brain areas, but no change in the level of
expression of aBgtx-nAChRs (Picciotto et al., 1995). A
large number of studies have used these mutated animals to
examine the role of b2-containing nAChRs in learning and
memory, neurodegeneration, drug reinforcement, nocicep-
tion, the development of the visual system and the
organisation of sleep.
5.3.1. Learning, memory and neuroprotection
Nicotinic transmission participates in many cognitive
processes. Nic improves performances in various tasks
involving spatial and associative learning, working memory
and attention, whereas mecamylamine (Mec) (a general
nicotinic antagonist) impairs memory performance (Levin,
2002). Behavioural tests have shown that b2 Ko mice do not
have the Nic–induced enhancement of passive avoidance
performance that is thought to model learning and memory
(Picciotto et al., 1995). Additionally, young b2 Ko mice
perform identically to WT mice in the Morris water maze
test, a cognitive test that assesses the acquisition of spatial
information, but 22–24 month old b2 Ko mice show a
significant deficit that indicates a major impairment in
spatial memory (Zoli et al., 1999). Histological analyses of
the brain of these aged b2 Ko mice show region-specific
cerebral cortex alterations with neocortical hypotrophy, a
loss of hippocampal neurons, and astro- and micro-gliosis.
b2 Ko mice also have increased cortical susceptibility to
ibotenic acid lesions, and primary cortical neurons obtained
from them no longer have Nic-induced neuroprotection
(Laudenbach et al., 2002).
These studies suggest that b2-containing receptors
contribute to neuronal survival and the maintenance of
cognitive performance during aging.
5.3.2. Drug reinforcement
It is believed that the reinforcement properties of Nic are
due to its capacity to modulate the dopaminergic system.
Like many drugs of abuse, Nic exerts its additive properties
by increasing DA release in the ventral part of the
mesostriatal dopaminergic pathway. This effect is partially
due to the direct activation of NAChRs on the dopaminergic
neurons of the VTA, because the direct infusion of nicotinic
antagonists into the VTApreventsNic-elicited DA release in
the nucleus accumbens, and blocks systemic Nic self-
administration in rats (Corrigall et al., 1994; Picciotto and
the terminal field of mesolimbic neurons, but the direct
administration of nicotinic antagonists on the nucleus
accumbens does not block the self-administration of Nic.
Experiments performed in b2 Ko mice have shown that
somatic b2-containing receptors are required for the effects
of Nic on the firing rate of DA neurons invitro as well as for
the release of DA from striatal synaptosomes (Champtiaux
et al., 2003; Picciotto et al., 1998). Furthermore, b2 Ko mice
do not learn to self-administer Nic even if they have learnt to
self-administer cocaine and WT mice self-administer both
drugs (Picciotto et al., 1998). Overall, these studies reveal
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396 379
that b2-containing nAChRs located in dopaminergic
neurons are important for the regulation of DA and, by
doing so, they are also important for mediating the addictive
properties of Nic (see also Section 2.3).
5.3.3. Development of the nervous system
nAChRs are expressed very early in the nervous system,
where they are not only finely regulated during CNS
development but probably actively contribute to it. In the
visual system of mammals, the refinement of the formation
of eye-specific layers at thalamic level depends on retinal
waves of spontaneous activity that rely on nAChR
activation (Rossi et al., 2001; Champtiaux and Changeux,
2002). Between P1 and P10, spontaneous retinal waves are
mediated by nAChRs and are sensitive to nicotinic
antagonist blockade but, at about P10, the waves become
insensitive to nicotinic antagonists and sensitive to
glutamatergic antagonists (Bansal et al., 2000). b2 Ko
mice have retinal waves with altered spatiotemporal
properties and retinofugal projections in the dorsolateral
geniculate nucleus (dLGL) and superior colliculus that do
not segregate into eye-specific areas (Rossi et al., 2001).
Furthermore, recent anatomical and functional studies of
dLGL b2 Ko mice have revealed normal gross retinotopy
but disrupted fine mapping, a loss of retinotopicity in the
nasoventral visual axis, and a gain in on/off cell
organisation (Grubb et al., 2003). b2 Ko mice also have
reduced visual acuity and functional expansion of the
binocular subfield of the primary visual cortex (Rossi et al.,
b2-containing receptors are necessary for the normal
development of the visual system, but the a subunit that
coassembles with the b2 subunit to mediate these effects is
unknown: the pattern of retinothalamic projection is
normal in a4 and a6 Ko mice (two subunits highly
expressed in the visual system) and, in a3 Ko mice, retinal
waves are not abolished although have altered spatiotem-
poral characteristics (reviewed in Champtiaux and Chan-
These visual pathway studies suggest that adequate
nAChR activation in other brain areas during development
may be essential for the anatomical and functional
maturation of cerebral neuronal circuits.
5.3.4. Organisation of sleep
There is evidence in humans that mutations in a4 and b2
subunits are correlated with seizures occurring during slow-
wave sleep. The use of b2 Ko mice has made it possible to
demonstrate that nAChRs containing the b2 subunit can
influence the REM sleep, controlling the duration and onset
of REM sleep episodes and the REM sleep-promoting
effects ofstress(Cohenetal.,2002;Lenaetal., 2004).These
findings, and the fact that b2-containing receptors are
important in controlling the rhythms of breathing and
arousal during sleep, should be kept in mind especially in
newborns, who are at risk of sudden infant death syndrome
and in whom correct REM sleep can influence the forming
neuronal circuits. This is a further stimulus to avoid the
exposure of newborns to passive smoking.
5.4. Phenotype of a6 and b3 Ko mice
In the CNS, the a6 and b3 subunits colocalise in
dopaminergic neurons and retina (Champtiaux et al., 2002;
Moretti et al., 2004). Deletion of the a6 subunit does not
change the level of mRNA for the a3–a5, a7, b2 and b4
subunits in the different brain regions (Champtiaux et al.,
2002) and, in b3 Ko mice, no changes in the mRNA level for
these and the a6 subunits have been found in the substantia
nigra (SN) and ventral tegmental area (VTA) (Cui et al.,
2003). In both a6 and b3 Ko mice, the major finding is a
global loss of high affinity aCntxMII binding in striatal
nerve terminals and, in a6 Ko mice, there is also the
disappearance of aCntxMII binding sites from retina, optic
nerve and the retina-target tissues of the superior colliculus
and dLGL (Champtiaux and Changeux, 2002). The absence
of a6-containing receptors in a6 Ko mice does not alter
the anatomy of the dopaminergic (Champtiaux and
Changeux, 2002; Cui et al., 2003) and visual pathways,
and the organisation of the retinothalamic projections is
Biochemical and pharmacological studies of striatal
extracts from WTand a6 Ko mice have identified two major
population of nAChRs: a4b2*and a6b2*(all of which are
involved in DA release from synaptosomes, Champtiaux
et al., 2003). A subset of about 40% of the a6b2*nAChRs in
rat and WT mouse striatum (Champtiaux et al., 2003) also
contain the a4 subunit, and competition binding experi-
ments on immunoimmobilised a6 subtypes using aCtxMII
have shown that the toxin biphasically displaced Epi with
both high and low affinity (Fig. 4).
nAChRs obtained from a4?/?striatum (which mainly
contains the a6 and b2 subunits) have shown that aCtxMII
displacement of Epi binding is only monophasic with a
single high-affinity site (Fig. 4). These pharmacological
results clearly indicate that, in a fraction of WT striatal a6*
receptors, one of the two Epi binding sites (located at the
interface between the a and b subunits) is made up of an
a4b2interfacewith alowaffinity foraCtxMII. Thisbinding
site is absent from the a6 receptors present in the striatum of
a4 Ko mice, which therefore have both binding sites with
high affinity for the toxin (Fig. 4).
Electrophysiological studies of DA neurons of WT, a4,
a6, and double a4/a6 Ko mice indicate that the major
heteromeric functional subtype expressed in dopaminergic
soma is a4b2, which is probably the subtype that
contributes to Nic reinforcement (Champtiaux et al.,
a6–purified receptor, and the lack of aCtxMII binding sites
and sensitivity in the striatum of b3Ko mice, indicate that
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396380
both the a6 and b3 subunits coexist in the same receptor in
the large majority of aCtxMII-sensitive receptors that
modulate DA release from striatal nerve terminals.
a6-containing receptors do not seem to be involved in
the DA release induced by systemic Nic because in vivo
microdialysis studies of freely moving mice have shown
that there is no difference in DA levels in the ventral
striatum of WTand Ko mice under basal condition or after
systemic Nic injection. However, as binding studies with
3H-Epi have revealed an increase in a4b2 receptors in the
SN-VTA, of a6 Ko mice, a functional compensation by
these receptors cannot be excluded (Champtiaux et al.,
a6 Ko mice do not show a change in locomotor activity,
but b3 Ko mice also display increased locomotor activity in
the open field area and reduced prepulse inhibition of the
acoustic startle response, two behaviours that are partially
controlled by nigrostriatal and mesolimbic dopaminergic
containing the b3 subunit may also modulate the
dopaminergic pathways that control these two important
5.5. Phenotype of a7 Ko mice
a7 Ko mice are viable and their brain anatomy is
apparently normal, but a more detailed search for specific
phenotypes is under way. The hallmark of these mice is the
loss of aBgtx receptors and the lack of Nic-evoked fast
desensitising currents in neurons (Orr-Urtreger et al.,
At high doses, Nic causes seizures and previous studies
have suggested that sensitivity to Nic-induced seizures
may be related to the density of aBgtx nAChRs but,
surprisingly, there is no difference in the dose of Nic
necessary to induce seizures in a7 Ko mice (Franceschini
et al., 2002). However, findings in another mouse model
expressing the ‘‘knock in’’ L250T mutation, a leucine to
threonine mutation in the transmembrane M2 region, that
results in a slow desensitizing receptor, suggest that the a7
receptor may play a role in Nic-induced seizures (Broide
et al., 2002). Mice homozygous for the mutation have a
lethal phenotype, whereas heterozygous animals survive
but have altered a7- type currents with increased
amplitudes and slower desensitisation. In comparison
with control mice, the heterozygous mice experienced a
significantly greater number of generalised tonic clonic
seizures in response to Nic. Homozygous L250T mutant
mice show increased apoptosis in the somatosensory
cortex, probably due to increased Ca2+influx through the
non-desensitising L250T a7 receptors. These data suggest
that aBgtx-nAChR activation may play a role in cell
death and that, by stimulating apoptosis, they may also
influence development. This is also suggested by the
finding that the antagonist aBgtx rescues ciliary ganglion
neurons that physiologically undergo apoptosis (Renshaw
et al., 1993). Moreover, in an experimental model of
anoxia based on ibotenate-induced cortical lesions,
newborn a7 Ko mice have smaller lesions than WT
controls (Laudenbach et al., 2002), thus suggesting that
a7 nAChRs may be important in regulating neuronal
survival (see 1.2.2).
The role of a7-containing receptors in the peripheral
nervous system has also been studied. In vivo and in
vitro studies have shown that a7 receptors are not important
for parasympathetic-mediated responses as both the nega-
tive inotropic effect of Nic and the baroreflex-mediated
parasympathetic responses to vasoconstriction are unchan-
ged in Ko mice (Franceschini et al., 2000). On the contrary,
a7 receptors contribute to the sympathetic response:
the increased heart rate, following baroreflex-mediated
activation of the sympathetic nerve, is consistently reduced
in a7 Ko mice, and this is not due to impaired availability of
NA in sympathetic nerve terminals (Franceschini et al.,
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396381
Fig. 4. Displacement of125I-Epibatine binding by aCntxMII in immu-
noimmobilised subtypes. The a4b2 subtype from a6KO (5), the a6b2b3
a4Ko mice (&) were incubated with increasing concentrations of aCntx-
MII. The inhibition curve of aCntxMII is biphasic on the WT a6 subtype
(Ki 1.54 nM and >10000 nM) and monophasicon the a4Ko a6 subtype (Ki
1.07 nM). These pharmacological results indicate that in a fraction of WT
striatal a6*receptors, one of the two Epi binding sites (located at the
interface between the a and b subunits) is made up of an a4b2 interface
with low affinity for aCtxMII. This binding site is absent from the a6
receptors present in the striatum of a4 Ko mice, which therefore have both
binding sites with high affinity for the toxins. From these data and the data
from immmunprecipitation experiments, we believe that the possible
arrangement of the subunit in the different subtypes are those indicated
by the scheme.
5.6. Phenotype of a9 Ko mice
The a9 subunit is not expressed in the brain, but is
expressed in cochlear hair cells innervated by cholinergic
efferent fibres originating from the brainstem/superior
olivary complex. a9 Ko mice show abnormal development
of the synaptic connections in cochlear outer hair cells and
abnormal coclear responses after efferent fibre activation,
thus suggesting that a9-containing receptors play a role in
this cholinergic system (Elgoyhen et al., 1994).
Ko mice experiments have shown that brain NAChRs are
not essential for survival or the execution of basic
behaviours: for example, b2 and a4 Ko mice, which
completely lack the most abundant high-affinity nAChRs,
can accomplish routine behaviour. However, NAChRs are
important for the fine control of a number of more
sophisticated and complex behaviours that can be evaluated
only by means of appropriate tests or in particularly labile
situations such as the aged brain.
These findings put NAChRs in a different and perhaps
more important perspective in terms of their involvement in
brain pathologies and as drug targets. Many pathological
situations involve a lack of fine control and tuning rather
than complete loss of a particular function, and the
pharmacological restoration of appropriate tuning may have
a very important clinical effect.
6. NAChRs in pathology
Neuronal NAChRs are involved in a wide variety of
diseases affecting the nervous system and non-neuronal
tissues. We here review the diseases in which NAChR
involvement has been experimentally validated.
6.1. Diseases affecting the nervous system
6.1.1. Age dependent disorders
126.96.36.199. Tourette syndrome. Is a chronic, familial neurop-
sychiatric disorder involving persistent extrapyramidal
movement disturbances, inappropriate vocalisations and
cacolalia.Itiscommonlytreated with neuroleptics,butthese
are not always effective and can have toxic effects. The
administration of Nic in the form of chewing gum or a
transdermal patch significantly improves the motor disorder
neuroleptics (Dursun et al., 1994; McConville et al., 1991,
1992; Sanberg et al., 1997; Silver et al., 1995, 2001; Honson
et al., 2004). Although there is as yet no evidence of the
effect of Nic administration suggests that they may play a
role in symptom manifestation, perhaps by modulating DA
release from striatal and limbic cortical areas.
188.8.131.52. Autism. Is a severe developmental disorder that
becomes apparent in earliest childhood and is characterised
by severely impaired social relations and communication,
planning and attention, and by restrictive, odd and
stereotyped behaviour. There is no drug that specifically
targets autism. A number of genetic and brain biochemical,
neurochemical and morphological features have been
reported in these patients, but there is no conclusive
evidence concerning the etiopathogenesis of the disease.
Abnormalities in the brain cholinergic system have recently
been reported (Perry et al., 2001), including the fact that
there is a decreased number of a4b2 nAChRs in the parietal
cortex and cerebellum and that a7 levels remain normal in
cortex but increase in the cerebellum (Lee et al., 2002;
Martin-Ruiz et al., 2004). Moreover studies on b2 Ko mice
have shown that these animals have altered social behaviour
resembling those present in autism and attention deficit
hyperactivity disorder (Granon et al., 2003). These findings
can explain some of the neurological dysfunctions in these
patients (e.g. cerebellar abnormalities) and provide some
insights into the cellular and molecular mechanisms of the
abnormal brain development, thus opening up possible new
therapeutic approaches towards controlling at least some of
the nicotine-controlled brain functions.
184.108.40.206. Attention-deficit hyperactivity disorder (ADHD). Is
an inheritable multigenic psychiatric disorder of childhood
characterised by difficulties in attending to tasks, hyper-
activity and hyperactivity-impulsive symptoms. Several
findings suggest that NAChRs are involved in this disease:
ADHD is associated with early smoking, maternal smoking
is a risk factor (Leonard et al., 2001), nicotinic drugs have
positive effects on experimental ADHD, and Nic increases
DA release (it is known that an important drug treatment for
the disease inhibits the DA transporter and increases
dopaminergic activity). However, no genetic evidence of
NAChR involvement has been reported, and a search for an
association between ADHD and polymorphisms in human
a4 and a7 genes was unsuccessful (Kent et al., 2001; Todd
et al., 2003).
220.127.116.11. Schizophrenia. The
NAChRs in schizophrenia is suggested by: (1) the high
prevalence of smoking among schizophrenic patients (90%
comparedto 33% in thegeneralpopulation (Lohr and Flynn,
1992; Perry et al., 2001; Poirier et al., 2002); (2) the fact that
neuroleptic neuronal side effects are fewer among smokers
(Poirier et al., 2002); and (3) the fact that there is a positive
correlation between smoking and negative (but not positive)
symptoms (Patkar et al., 2002). Recent brain autopsy data
indicate that schizophrenic patients have altered NAChRs
(Court et al., 2000; Martin-Ruiz et al., 2003). The
involvement of heteromeric receptors is doubtful. Unlike
preliminary studies, recent investigations evaluating the
confounding effects of smoking using more appropriate
techniques have failed to show any differences between
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396 382
controls and schizophrenic patients in terms of3H-Nic and
3H-Epi binding (Breese et al., 2000) and the levels of a4, a3
or b2 subunits in the hippocampus, thalamus, cortex and
caudate (Martin-Ruiz et al., 2003). All of these data suggest
that the most diffuse heteromeric receptors in brain are not
modified in schizophrenia, although it is possible that heavy
smoking (whose effects on the patients’ brains are not
completely known) may mask some small modifications.
On the other hand, the involvement of homomeric a7
receptors is much more relevant. Schizophrenic patients
have a small, but significant and reproducible decrease in
125I-aBgtx binding sites and a7 immunoreactivity in the
hippocampus (Freedman et al., 1995; Leonard et al., 1996),
the reticular nucleus of the thalamus, and the cingulated and
frontal cortex (Martin-Ruiz et al., 2003). Genetic linkage
receptors. The P50 auditory evoked potential gating deficit
that is observedin some schizophrenic patient families maps
to a region of chromosome 15 q13–14 (Leonard et al., 1996)
2001). The human gene is partially duplicated in schizo-
phrenic families (Freedman et al., 2001; Xu et al., 2001) and
genetic studies suggest that this chromosomal location is
involved in the genetic transmission of schizophrenia
(Freedman et al., 2001). Polymorphisms in the core
promoter of the human a7 gene are associated with
schizophrenia and with diminished inhibition of the P50
response (Freedman et al., 2003; Leonard et al., 2002). The
finding that Nic transiently reverses P50 deficit is consistent
with the fact that a7 receptors may control auditory sensory
gating (see Leonard et al. (1996)).
Altogether, these data indicate that schizophrenics may
have an a7 receptor deficit that they attempt to overcome by
smoking. More clinical, genetic and experimental investiga-
tions are clearly needed but, in the meantime, nicotinic drug
treatment in schizophrenia could be worth exploring.
6.1.2. Age-independent disorders
18.104.22.168. Epilepsy and febrile convulsions. Recent genetic
studies have demonstrated the central role of ion channels in
the pathophysiology of idiopathic epilepsies (Scheffer and
Berkovic, 2003). Autosomal dominant nocturnal frontal
lobe epilepsy (a partial epilepsy that causes clusters of brief,
frequent and violent seizures during sleep) is a genetic
diseasewith abnormalities located in chromosome 20q13.2–
q13.3, which contains the gene encoding the a4 nicotinic
subunit, the most commonly expressed subunit in human
brain (Phillips et al., 1995). Mutations in the a4 gene have
been described in families from many countries and they are
located in hotspots along the transmembrane domain that
forms the ion channel (Hirose et al., 1999; McLellan et al.,
2003; Saenz et al., 1999; Steinlein et al., 1995, 1997).
Mutations in the gene CHRNB2, which encodes the b2
nicotinic subunit, have also been described and are again
localised in regions that form the ion channel (De Fusco
et al., 2000; Phillips et al., 2001). The mutations lead to
increased sensitivity to ACh and perhaps a change in Ca2+
permeability, thus facilitating the synchronisation of the
spontaneous oscillations in the thalamo-cortical circuits and
generating seizures (see Raggenbass and Bertrand (2002)).
Further evidenceof the relevance ofa4-containingreceptors
in epilepsy comes from the finding that mice lacking a4
subunits are more sensitive to the proconvulsant effects of
GABA antagonists (McColl et al., 2003) and the fact that
antinicotinic drugs haveantiepilectic activity(Loscheretal.,
2003). However, the mutations in NAChRs do not cover all
of the symptoms of ADNFLE and probably represent only
of the disease (Sutor and Zolles, 2001).
It has been reported that there is an association between
the CHRNA4 gene and febrile convulsions in children
(Sahakian et al., 1989), but not with any b2 subunit gene
polymorphism (Peng et al., 2004). In the case of febrile
convulsions, a4 receptor abnormalities could modify not
only neuronal circuitry, but also the permeability of the
blood–brain barrier that is already affected by the high
There are experimental indications that a7 receptors are
also involved in seizure control: one strain of mice with a
large number of brain
prone to develop seizures in response to Nic (Marks et al.,
1989), and a correlation exists between the level of
NAChRs in hippocampal astrocytes and the susceptibility
of mice strains to Nic seizures (Gahring et al., 2004a) [see
also 5.5]. Although nicotinic abnormalities have been
reported in a very small minority of epilepsies, these data
confirm that both heteromeric and homomeric nicotinic
receptor subtypes are important in the control of brain
excitability, and that appropriate nicotinic agents could be
of value in seizure control or prevention in at least some
forms of epilepsy.
125I-aBgtx binding sites is more
22.214.171.124. Depression and anxiety. Although to a lesser
extent than schizophrenic patients, there is evidence that
the prevalence of tobacco smoking is higher in depressed
individuals than in the normal population (Poirier et al.,
2002), smoking cessation is associated with depression in
individuals with a history of depression, Nic has been
reported to be antidepressive and mood stabiliser in humans
(Shytle et al., 2002a,b), and a number of antidepressants are
antinicotinic agents (Fryer and Lukas, 1999a,b). NAChRs
have been involved in both the anxiolytic and anxiogenic
effects of Nic in experimental animals (see review by
Picciotto et al. (2002)). Studies suggest that NAChRs can
modulate the nervous pathways related to depression and
anxiety, but the results are too preliminary to enable us to
6.1.3. Age-related degenerative diseases of the brain
Alzheimer’s disease (AD) and Parkinson’s disease are
psychiatric or neurological degenerative disorders in which
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396383
cholinergic pathways are consistently affected. The nucleus
basalis of Meynert undergoes varying degrees of degenera-
tion and cortical choline acetyltransferase activity is
consistently decreased (see Clementi et al. (2000) for a
review). Early evidence that NAChRs are involved in these
diseases came from epidemiological data correlating
smoking and AD and PD. Careful epidemiological studies
confirmed that tobacco smoking reduces the risk of
developing PD and that this relationship is not due to any
obvious confounding factors (Clementi et al., 2000). On the
other hand, the protective effect of tobacco smoking in AD
remains highly controversial (Kukull, 2001). A number of
reasons may explain the differences in the protective effects
of tobacco smoking between PD and AD, one of which may
be that there is a vascular component in AD (but less in PD),
and it is known that smoking adversely affects cardiovas-
cular and cerebrovascular function.
126.96.36.199. Parkinson’sdisease. Inadditiontotheinvolvement
of the nicotinic cholinergic system (Burghaus et al., 2003)
the relevant pathology of this disease is the loss of
dopaminergic neurons in the nigro-striatal pathway.
NAChRs are very important in promoting the release of
DA in this pathway, and both the a6b3b2 and the a4b2
subtype in striatum are responsible for the release of
DA from nerve endings (Champtiaux et al., 2003; Quik
and Kulak, 2002). Other neuronl systems that contribute
to the proper function of the nigro-striatal pathway
contain NAChRs (for example, a7 on glutamatergic
nerve endings, a4b2 on cholinergic and gabaergic
interneurons, a6a4b2 on GABAergic neurons of the
SN) (see Section 2.3).
In experimental rodent and monkey models of PD, there
is a selective decrease in the number of a6-, a4- and b2-
containing receptors as detected by means of specific ligand
binding or immunoprecipitation experiments (Zoli et al.,
2002a,b; Champtiaux et al., 2003; Kulak et al., 2002; Lai
et al., 2004; Quik et al., 2003, 2004; Quik and Kulak, 2002).
There isageneralconsensus that3H-Nic and3H-Epibinding
is decreased in the striatum of PD patients (Court et al.,
2000; Guan et al., 2002; Martin-Ruiz et al., 2000; Quik and
Kulak, 2002), and we have recently seen a consistent
decrease in the a6 subunit in both human and experimental
models of PD (Gotti et al., unpublished data). The
importance of a6-containing receptors in PD is also
supported by the experiments of Lai et al (Lai et al.,
2004). This group reports that in an experimental PD
monkeys, a selective reversal of the a6-containing receptor
decrease is observed when monkeys recover from the
induced lesion. A decrease in nAChRs similar to that
reported in AD has also been found in the cerebral cortex
of PD patients, and is mainly due to a decrease in a4- and
a7-containing receptors (Burghauset al., 2003). These latter
findings are in agreement with the impaired cognitive
functions of some PD patients. All of the data briefly
disease and particularly the a6- and a4-containing receptors
in DA release from the nigrostriatal pathway, and the a4b2
subtype in the cognitive cortical aspects.
controversial results, with little or no improvement in
cognitive and motor symptoms (Kelton et al., 2000). These
results are not unexpected as a treatment that affects all
NAChRs,andnota selectivesubtype, unpredictably disrupts
the complex cholinergic network of the mesostriatal
dopaminergic pathway, and its positive effects can be
masked or counterbalanced by the activation of other
188.8.131.52. Alzheimer’s disease. There is general consensus
that the number of brain NAChRs is decreased in this
neurons (Clementi et al., 2000; Court et al., 2001; Nordberg,
1992, 1995; Nordberg et al., 1990; Schroder et al., 1991).
The most affected areas are the neocortical areas,
hippocampus, presubiculum and various thalamic nuclei.
It has more recently been shown that a4 nicotinic subunits
are decreased in cortical areas and in the hippocampus
(Court et al., 2000) which suggests that the loss of high
affinity agonist binding is due to the loss of a4-containing
receptors. The observation that the b2 subunit (which,
in brain) is not affected (Court et al., 2000; Engidawork
et al., 2001) is puzzling and difficult to explain (also see the
data on aging in b2 Ko mice (Picciotto and Zoli, 2002). A
relationship between NAChRs and AD also emerges from
genetic studies that show the presence of genetic poly-
morphisms of the neuronal a4 and b2 genes in some AD
patients (Cook et al., 2004; Kawamata and Shimohama,
The data concerning a7 receptors are more contradictory
and less convincing, and probably reflect unrecognised
differences among subgroups of patients or an imprecise
analysis of the cell distribution of the receptors (Court et al.,
2000; Wevers et al., 2000).
Another factor relating AD and NAChRs is b-amyloid
protein (b-AP), whose deposition is a typical feature of AD.
There is general consensus that b-AP plaques are related to
the neurodegeneration; b-AP is severely neurotoxic in vitro
low concentrations; in a mouse model of human AD, there is
an up-regulation of NAChRs, probably due to compensatory
mechanisms in response to b-AP burden (Bednar et al.,
2002) although the mechanisms of this effect are not clear
(see reviews by Clementi et al. (2000); Zamani and Allen
b-AP interacts with NAChRs at nM concentration with
the a4b2 subtype and at pM with the a7 receptors (Wang
et al., 2000a,b) and inhibits in a non-competitive way the
on a7 receptor is controversial since both the inhibition and
activation of wildtype and heterologously expressed a7
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396 384
receptors (Grassi et al., 2003; Liu et al., 2001; Pettit et al.,
2001)(Dineleyetal.,2002)hasbeen reported.The effects of
b-AP on both classes of NAChRs could contribute to the
impairments of memory and cognition in AD patients. As it
has also been shown that both the b2 and a7-containing
receptors contribute to neuronal survival during aging
(Laudenbach et al., 2002; Zoli et al., 1999), it is possible that
the effects of b-AP on these receptors may be responsible of
a premature neuronal death.
The important involvement of NAChRs in AD suggests
We can envisage two objectives for nicotinic therapy: the
slowdown of disease development and symptomatic
relief. The marked regional variations in nicotinic receptor
densities, as well as their specific reductions in the absence
of a general decrease in the number of neurons ((Schroder
et al., 1991), also see above), suggest that receptor
abnormalities may occuratanearlystageofthe pathological
process before irreversible neuronal degeneration takes
place, and that they may be due to a neurotoxic effect of
b-AP. At later stages, microvascular abnormalities may
account for the severe neuronal loss and precipitate the
clinical symptoms. Early treatment with nicotinic agonists
or AChE inhibitors could therefore be positive, and the
epidemiological data relating heavy smoking in AD patients
with this suggestion. However, it is not yet clear whether
it is better to intervene with a drug affecting all NAChRs
(nicotine or AChE inhibitors) or a subtype-selective drug, in
which case it would be necessary to identify the most
appropriate receptor subtype. Preliminary clinical data
indicate that a nicotinic approach may be feasible, although
the therapeutic effects of cholinergic agents are not
dramatic. In AD patients, Nic improves perceptual and
visual attention deficits (Jones et al., 1992) attention
performances (Rezvani and Levin, 2001) and semantic
memory performance and these effects persist over time. Of
the cholinesterase inhibitors, tacrine (Knopman et al., 1996)
and galanthamine (which are also channel modulators) have
beneficial effects in some group of patients, Nootropic
drugs, which have some place in AD treatment, potentiate
a4b2 activity (Zhao et al., 2001).
6.2. Pathologies in non-neuronal tissues and cells
6.2.1. Lung cells
NAChRs have been implicated in a number of diseases
characterised by cell proliferation on the basis of the results
of epidemiological studies linking tobacco smoking to lung
184.108.40.206. Small-cell lung carcinoma. Lung cancer is one of
the major causes of death and is associated with exposure to
tobacco smoke (Am. Cancer Soc., 1994). Small-cell lung
carcinomas (SCLC) are particularly linked to smoking
(Weiss and Benarde, 1983). Among the more than 2000
smoke constituents, Nic and related alkaloids and a
none (NNK), which can bind and stimulate nAChRs
(Crooks and Dwoskin, 1997), may contribute to this effect.
SCLC cell lines have binding sites for aBgtx (Cattaneo
et al., 1993; Maneckjee and Minna, 1990; Quik et al., 1994;
Schuller and Orloff, 1998), for a-CntxMII (Codignola et al.,
1996) and also express mRNA for the a2–a5, a7, b2 and b4
subunits (Chini et al., 1992; Tarroni et al., 1992; West et al.,
2003). These cell lines synthesise and release ACh (Song
et al., 2003), thus suggesting that there is an autocrine or
paracrine cholinergic pathway in lung tumours. Acute and
long-term treatment of SCLC cell lines invitro with Nic and
NNK stimulate proliferation (Cattaneo et al., 1993; Fucile
et al., 1997), an effect that depends on Ca2+influx and is
suppressed by aBgtx (Codignola et al., 1994; Maneckjee
and Minna, 1990). These data suggest that NAChRs control
the rate of proliferation of these cell lines, probably via a
Ca2+influx that activates MAP and Akt kinases (Codignola
et al., 1994; Cattaneo et al., 1997; West et al., 2003; Schuller
et al., 2000). Which receptor subtype is involved is not yet
clear, but experiments with selective nicotinic antagonists
suggest that Akt kinase activation is via an a4b2 receptor,
whereas MAP kinase activation is probably via an a7
receptor (Minna, 2003; West et al., 2003).
220.127.116.11. Other lung cells. NAChRs have been found in
human and murine bronchial epithelial cells. These cells
express a3, a5, b2, b4 and a7 receptor subunits that form
functional ion channels that are highly permeable to Ca2+
(Maus et al., 1998; Zia et al., 1997). Stimulation by Nic
induces the cell release of granulocyte-macrophage colony
stimulating factor (Klapproth et al., 1998). Smoking induces
in vitro, and a long-lasting increase in intracellular Ca2+
concentration. The high levels of Nic in lung can activate a7
receptors in lung macrophages, thus inhibiting the release of
probably play an important role in controlling hormone and
mucus secretion in the bronchi, in cell-to-cell communica-
tion, adhesion and tactility, and in ciliary motion. The data
indicate that long exposure to high Nic concentrations, as in
chronic smokers, plays an important role in lung cancers,
bronchial toxicity and the control of local immune
6.3. Vascular smooth muscle and endothelial cells
Tobacco smoking increases the risk of vascular occlusion
after bypass grafting and angioplasty, and the failure of
vascular grafts due to intima hyperplasia caused by smooth
muscle cell proliferation (Crooks and Dwoskin, 1997). Very
low Nic concentrations (10?8M, similar to those found in
the blood of smokers) can enhance DNA synthesis and
stimulate endothelial cell proliferation in vitro (Carty et al.,
1997; Villablanca, 1998). Long-term exposure to Nic
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396385
accelerates intimal hyperplasia after endothelial lesion
(Hamasaki et al., 1997). Furthermore, together with high
cholesterollevels,smokingis a risk factor in atherosclerosis,
and chronic Nic associated with a cholesterol diet increases
vascular plaque formation in rabbits (Strohschneider et al.,
1994). Nic may induce smooth muscle cell proliferation by
activating the MAP kinase pathways, releasing mitogenic
factors such as the basic fibroblast growth factor, and
stimulating the metalloproteases that are important in cell
migration (Carty et al., 1996).
6.4. Dysautonomia and blood pressure control
The control of blood pressure by peripheral autonomic
ganglia is a well-established physiological fact. Peripheral
autonomic neuropathies lead to neurogenic orthostatic
hypotension and other signs of ganglionic failure. In a
consistent number of patients, autonomic neuropathy can be
due to autoimmune phenomena as their blood contains Abs
against ganglionic receptors (Balestra et al., 2000a; Gold-
stein et al., 2002; Klein et al., 2003; Sandroni et al., 2004;
Vernino et al., 2000). Immunisation with a3 ganglionic
subunit in rabbits induces an experimental autoimmune
autonomicdiseasewith symptomssimilar tothose present in
patients with autonomic neuropathy (Lennon et al., 2003;
Verninoetal., 2004).Furthermoresomesera ofpatients with
autoimmune autonomic neuropathy can induce a mild form
of this syndrome in mice (Vernino et al., 2004). Anti-
neuronal Abs are also present in patients with SCLC, and
account for various paraneoplastic symptoms and, in
et al., 1991; Gotti et al., 2001). These Abs are useful
markers for diagnostic purposes, and can partially explain
the pathophysiology of the disease, in a way similar to that
found in myasthenia gravis (Drachman, 2003) suggesting
that a selective removal of a3 Abs could be beneficial in this
In addition to the peripheral ganglionic control of blood
pressure, it is known that the circulation is also under central
cholinergic control. NAChRs are present in key areas
regulating blood pressure, such as the nucleus of the solitary
tract (Wada et al., 1989; Clarke, 1993), and evidence
suggests that nAChRs are involvedin the regulationofblood
pressure and hypertension (see Buccafusco (1996)). The
of blood pressure control relates to spontaneously hyper-
tensive rats (SHR). These animals spontaneously develop
hypertension in adulthood and have fewer nAChRs (a3 and
a4 subunits) than age-matched controls in the cerebral
cortex, thalamus, midbrain, medulla oblongata, and spinal
cord (Hernandez et al., 2003; Khan et al., 1996; Ueno et al.,
2002). The injection of Nic or cytisine intrathecally or in the
area postrema in adult SHR rats increases blood pressure,
tachycardia and nociception behaviour. Antihypertensive
pharmacological treatment does not modify the number of
is not due to a homeostatic response (Khan et al., 1994,
1996). Interestingly, these animals have a cognitive
impairment typical of NAChR loss, and could be a genetic
model for the study of dementia and ADHD (Gattu et al.,
1997a,b; Ueno et al., 2002).
6.5. Intestinal epithelium
Overwhelming epidemiological and clinical evidence
suggests that NAChRs are involved in inflammatory bowel
disease (Baron, 1996; Richardson et al., 2003). There is a
close negative association between smoking and ulcerative
colitis, but smokers are at increased risk of developing
Crohn’s disease (reviewed in (Thomas et al., 1998).
Smoking or Nic treatment have an adverse effect on the
clinical course of Crohn’s disease (Bonapace and Mays,
1997) whereas treatment with transdermal Nic patches
significantly improves ulcerative colitis in the active
phase, a benefit similar to that obtained with corticoster-
oids (Guslandi and Tittobello, 1996; Pullan et al.,
1994). However, a careful evaluation of long-term Nic
treatment is not available (Kennedy, 1996; Thomas et al.,
The mechanismsby which Nic havesuch opposite effects
concerning the decreased secretion of TNFa and proin-
flammatory cytotoxins by macrophages via a7 activation,
and the divergent effects of Nic on cytokine levels in colonic
mucosa (Eliakim and Karmeli, 2003), can add further light
on this matter (see also 4.3).
7. Conclusions and perspectives
Over the last few years, a number of important
technological advances have increased our understanding
of the functioning of NAChRs. In particular, the application
of new molecular and cellular techniques, immunological
assays with subunit-specific Abs for NAChR localisation
and purification, in vivo localisation using non-invasive
imaging techniques, new selective ligands, and especially
the availability of Ko and Kin animals for the individual
subunits, have made it possible to correlate the subunit
composition of NAChRs with specific nicotine-elicited
behaviours and better define some of the in vivo
physiological functions of the NAChR subtypes.
The most relevant new findings emerging from these
studies are the widespread expression of nAChRs, their
specific and complex organisation, and their relevance to
normal brain function. Moreover, the combination of
clinical research with the above approaches has better
defined the involvement of NAChRs in a growing number of
nervous pathologies other than degenerative diseases, such
as autism, ADHD, anxiety and schizophrenia, and led to
the discovery of NAChRs in non-neuronal cells and their
role in non-nervous diseases such as inflammatory reaction,
C. Gotti, F. Clementi/Progress in Neurobiology 74 (2004) 363–396386
pemphygus, pathological angiogenesis, tumour growth,
Unfortunately, except for the large family of toxins, the
compounds so far available are largely not subtype selective
and some can have agonist action on one subtype coupled
with antagonistic actions on others. Furthermore, although
several nicotinic drugs have reached clinical application for
pain, AD, PD, nicotine addiction, anxiety and La Tourette
syndrome, they never reached the final goal because of their
poor efficacy and/or toxic effects.
A number of reasons may explain this failure. First of all,
the widespread localisation of NAChRs that do not control a
single specific nervous pathway, but modulate several at a
time; secondly, the lack of reliable experimental models for
testing the possible usefulness of new drugs because of the
complexity of human brain diseases and the specific
localisations of animal and human NAChRs; thirdly, we
still do notknowifit ispreferable tousespecificdrug bullets
or drugs capable of interfering with different receptor
subtypes and, furthermore, in some pathological conditions
and antagonists are both positive), it is not clear whether the
receptors are decreased or desensitised or if it is better to
intervene with agonists, antagonists or channel modifiers;
fourthly, the most appropriate kinetics with which the
treatment should be applied is unknown. Nicotine, the only
after short intense pulses whereas it acts in heart even it is
administrated with a larger spectrum of drug delivery. Given
the different targets of nicotinic treatment, appropriate
kinetics could be relevant to increasing selectivity.
All of these uncertainties make it difficult to design
clinical trials but, without them, it is difficult to explore the
possible therapeutic applications of new compounds.
However, we strongly think that basic and preclinical
research on NAChRs should be strengthened because the
clinical application of this enormous pack of knowledgewill
certainly produce benefits for understanding and therapeu-
tically managing a discrete number of diseases of nervous
and non-nervous tissues.
The goal of current and future NAChR research is
therefore to continue combining our increasing knowledge
of native receptor structure and function, nicotinic ligand
docking sites and the genetic background of the behavioural
responses to nicotine in order to be able to design new
ligands specifically targeted against the subtypes and/or
mutated receptors at the origin of the disease states.
We are grateful to Drs. Jenny Court and Elaine Perry for
grants from the Italian MIUR (MM05152538) to Francesco
Clementi; from the European Research Training Network
HPRN-CT-2002-00258, the FISR-CNR Neurobiotecnologia
2003, the Fondazione Cariplo grant no. 2002/2010 to
F. Clementi; and from the FIRB (RBNE01RHZM) 2003 to
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