CONTENTS

 

 

 

OVELVIOW oo. eene eee ene eee e eee ened beeen t eee e basen 79
Peripheral Effects of Nicotine..................0cceeeeeeees 79
Central Sites of Nicotine Actions ......................005 80
Neuroendocrine Effects of Nicotine.....................55 81
Electrophysiological Effects of Nicotine ................. 81

Distribution and Cerebral Metabolic Effects of

Nicotine. ...... 0... cece cece eee eee e eee c tees eee enegaeeneneeas 82

Distribution of Nicotine ...............cccccece cece eenee eens 82
Tissue Distribution of Nicotine: Time Course

and Other Considerations .....................00008 82
Heterogeneity of Nicotine Uptake: Microauto-

radiographic and Subcellular Studies........... 85

Effects of Nicotine on Cerebral Metabolism ........... 85

Nicotine Receptors ............ccccccccc cece eee sees eee e ent eeeeentenes 88
Peripheral Nicotine Receptors...................c0sceeeaes 88
Radioligand Binding to Putative Nicotine Choliner-

gic Receptors in Mammalian Brain.................... 89
Agonist Binding................ccccceeeceeeeeseeseeuvaees 89
Radioligand Binding ...................ccceceeseeee eee 91
Antagonist Binding .................. cece cece ene e eee ees 91

Functional Significance of Nicotinic Binding Sites...92
High-Affinity Agonist Binding Sites ............... 92
Alpha-Bungarotoxin Binding Sites.................. 92
Behavioral and Physiological Studies.............. 93
The Neuroanatomical Distribution of Nicotinic
Binding Sites in the Brain...............0..ccccceeeeeee 93
High-Affinity Agonist Binding Sites............... 93
Rodent... 0... ccc cece cece cceeenneeeeeeennnnerneaneee 93
Monkey............ccccecee cence cece eeeeteeeneteseeeeeens 94
Human .... 2... cece een eee ee ee eeeeenaetennaeevens 94
Alpha-Bungarotoxin Binding Sites.................. 94
Molecular Biology ..............ccceeee eee eee eee e reece 95
Central Nicotinic Cholinergic Receptors: Pre- or

Postsynaptic?......... 00... c ccc cece cece eee tea eeeeneeetanees 95

Presynaptic Regulation of Neurotransmitter
Release.............cccce cece ce eceeeneeeenaeeteeseenneeees 95
Somatodendritic Postsynaptic Actions............. 95

77
 

Neuroendocrine and Endocrine Effects of Nicotine........ 95

 

 

 

 

Cholinergic Effects ..........000000ccccccseececeeceecceccceceen. 96
Modulation of Catecholamine and Serotonin
ACtivity oc ccccccceeccasecneessuusseneseeesessee. 97
Effects on Serotonergic Neurons.................... 99
Effects on Catecholaminergic Neurons.......... 100
Stimulation of Pituitary Hormones..................... 101
Arginine Vasopressin............6...0cccccceccecece. 102
The Pro-Opiomelanocorticotropin Group of
HOrmoness ............cccsccsscsecescassasseusenceccces 103
Thyroid... 0... ec cecceccesceeccceesersessseuecseeecceecee. 104
Adrenal Cortex ............0.cccccccseecccuescceueccenecceece. 104
Androgens ....... 0.0... eececcccsescseuececaesesansesuseccsccs 106
Estrogens .......... 00.0. cccceccecccuusececeeeeessueesscceeecccs, 106
Pancreas and Carbohydrate Metabolism .............. 107
Electrophysiological Actions of Nicotine.................... 107
Electrocortical Effects.........00....cccecccccceesccccececcn. 107
Spontaneous Electroencephalogram ..................... 108
Sensory Event-Related Potentials........................ 112
Cognitive Event-Related Potentials...................... 114
Motor Potentials. ..........00..00..ececcceeeccccescceeccccee. 115
Other Peripheral Effects Relevant to Tobacco Use...... 115
Psychophysiological Reactivity and Smoking......... 116
Psychophysiological Reactivity, Smoking Cessation,
and Relapse ....... 00... ......cccccsecccueeeeeuscceecececcs. 120
Summary and Conclusions.................ccccccssseeeececcc.., 123
References ......00.......ccccccecececcceeeeeeesccesecceeeeeccscc, 124

78
Overview

Nicotine, in tobacco smoking concentrations, is a powerful psy-
choactive drug (Domino 1973; Kumar and Lader 198]; Balfour 1984). A
wide variety of stimulant and depressant effects is observed in
animals and humans that involves the central and peripheral
nervous, cardiovascular, endocrine, gastrointestinal, and skeletal
motor systems. These heterogeneous effects, along with behavioral
and psychological variables, result in self-administration of tobacco,
tobacco dependence, and withdrawal phenomena with abrupt cessa-
tion of tobacco smoking. This Chapter discusses sites and mechan-
isms of nicotine actions that may help to explain why tobacco
products are self-administered.

The first Section of this Chapter provides general summaries of
several major effects of nicotine in the body. Following this broad
overview, the Chapter presents detailed discussions of sites and
mechanisms of nicotine action that may be particularly important to
understand tobacco use. Tissue distribution of nicotine, cerebral
metabolic effects, and nicotine receptor binding are reviewed. Next,
neuroendocrine and endocrine effects of nicotine are discussed.
Then, electrophysiological effects of nicotine are presented. Finally,
the effects of smoking on psychophysiological reactivity are discuss-
ed.

Peripheral Effects of Nicotine

Nicotine exerts its action on the cardiovascular, respiratory,
skeletal motor, and gastrointestinal systems through stimulation of
peripheral cholinergic neurons via afferent chemoreceptors and
ganglia of the autonomic nervous system (ANS) (Ginzel 1967b).
Inasmuch as both sympathetic and parasympathetic ganglia are
stimulated by levels of nicotine derived from tobacco smoking, the
end result depends on the summation of the effects of autonomic
ganglion stimulation and reflex effects. The resulting peripheral
physiological changes generally resemble sympathetic nervous sys-
tem (SNS) arousal, but there are also some effects of nicotine and
smoking that lead to physiological relaxation. For example, there is
usually an increase in heart rate and blood pressure immediately
following cigarette smoking. In addition, there is cutaneous vasocon-
striction of the distal extremities. In contrast, nicotine can relax
skeletal muscles (e.g., reduce patellar reflex) in humans and animals
via effects on Renshaw cells (Domino and Von Baumgarten 1969;
Ginzel and Eldred 1972; Ginzel 1987). But it also can enhance tension
in some muscles (e.g., trapezius muscle) (Fagerstrém and Gotestam
1977). Nicotine in small doses can enhance respiration through
stimulation of peripheral chemoreceptors. Yet, high nicotine doses
can cause respiratory failure. (See Appendix B for a discussion of

79
nicotine toxicity.) The gastrointestinal effects of nicotine are com-
plex, involving an increase in secretions and reduced motility for a
short period of time.

The peripheral actions of nicotine as a cholinergic agonist have
made it a valuable pharmacologic tool for studying nicotinic
cholinergic actions and functioning in many physiological systems.
This Chapter focuses on the mechanisms of nicotine’s actions
relevant to tobacco use. Several peripheral actions of nicotine, for
instance muscular relaxation, may contribute to the habitual use of
tobacco products (see smoking and stress in Chapter VI). However,
because the central nervous system (CNS) actions of nicotine and
resulting neurochemical and electrical effects mediate subsequent
biological and behavioral responses, a review of these actions
contributes to an understanding of the reinforcing effects of nicotine.

Central Sites of Nicotine Actions

Nicotinic binding sites or receptors in the brain have been
differentiated as very high, high, and low affinity types (Shimohama
et al. 1985; Sloan, Todd, Martin 1984; Sloan et al. 1985). In the rat
brain, when cholinergic muscarinic receptors are blocked, the
autoradiographic distribution of *H-acetylcholine (ACh) and *H-
nicotine are essentially identical (Clarke and Kumar 1984; Clarke,
Pert, Pert 1984). However, these brain binding sites differ from
peripheral nicotinic receptors in ganglia and skeletal muscle.

Chronic nicotine administration results in up-regulation in region-
al rat brain *H-ACh binding sites measured in the presence of
atropine to block the muscarinic sites (Schwartz and Kellar 1985).
Up-regulation of *H-nicotine binding sites also has been reported
after continuous nicotine infusions in mice (Marks, Burch, Collins
1983a). In contrast, most agonists that act on receptor sites in the
body, when given chronically, produce a reduction (or down-regula-
tion) in the number of receptors. Both Marks, Burch, and Collins
(1983b) and Schwartz and Kellar (1983, 1985) have suggested that
nicotinic cholinergic receptors undergo a functional blockade but
that sufficient recovery would allow enhanced behavioral responses
to low doses of nicotine to occur within 24 hr, as has been shown
behaviorally by Clarke and Kumar (1983) and Ksir and coworkers
(1985). This phenomenon may help to explain the tolerance to
nicotine that develops with repeated exposure. However, the time
course of changes in receptor number and other biological effects of
nicotine must be carefully compared to determine mechanisms
underlying tolerance. (See Chapter II for additional discussion.)

Several investigators have used in vitro autoradiography to
identify *H-nicotine binding sites in the rat brain. These audioradio-
graphic binding studies suggest where nicotine is acting. London,
Waller, and Wamsley (1985) have found the most intense localization

80
of ?H-labeled nicotine in the interpeduncular nucleus and medial
habenula.

Cerebral metabolism studies also suggest key sites of action.
London and colleagues (1985) have reported that nicotine stimulated
local cerebral glucose utilization (LCGU) by 139 percent over that of
the control in the medial habenula and by 50 to 100 percent in the
superior colliculus and the anteroventral thalamic and interpedun-
cular nuclei. Other areas of the brain showed moderate or no
significant changes. These effects of nicotine were blocked by
mecamylamine, a nicotinic receptor antagonist, confirming that they
acted via nicotinic receptors. Furthermore, they correlated well with
the distribution of *H-nicotine binding in the brain except in layer
IV of the neocortex, which showed nicotine binding but no change in
LCGU. Sites that show increased glucose utilization after nicotine
administration are probably functionally important loci of nicotinic
actions. When nicotine binding and increased energy utilization both
occur at a given site, it is likely to be involved in nicotine’s actions.

Neuroendocrine Effects of Nicotine

Some of the actions of nicotine result from the release of ACh and
other neurotransmitters, including norepinephrine (NE). Nicotinic
cholinergic agonists including nicotine, carbachol, and 1,l-dimethyl-4-
phenylpiperazinium (DMPP) release endogenous ACh from the
presynaptic cholinergic nerve terminals in addition to stimulating
postsynaptic nicotinic receptors (Chiou 1973; Chiou and Long 1969).
Nicotinic agonists also release ACh from rat cerebral cortical
synaptic vesicles and can release newly synthesized 3H-ACh from
synaptosomes prepared from the myenteric plexus of guinea pig
ileum and from mouse cortical synapses (Briggs and Cooper 1982;
Rowell and Winkler 1984). These effects are Ca?+-dependent and are
blocked by hexamethonium, a quarternary nicotinic receptor antago-
nist. In addition, nicotine-induced release of ACh in the hippocampal
synaptosomes is blocked by the ion channel blocker, histrionicotoxin
(Rapier et al. 1987). There is good evidence that nicotine releases
ACh by a presynaptic mechanism. In contrast, presynaptic musca-
rinic receptors, mostly of the M,-subtype, inhibit ACh release.
Nicotine administration increases the amounts of other chemicals in
the blood and brain, including serotonin, endogenous opioid peptides,
pituitary hormones, catecholamines, and vasopressin (Domino 1979;
Gilman et al. 1985; Marty and colleagues 1985). These chemicals may
be involved in reinforcing effects of nicotine (see Chapters IV, VI).

Electrophysiological Effects of Nicotine

Nicotine administration is accompanied by brain wave or electro-
encephalogram (EEG) activation in animals (Domino 1967). The EEG-
activating effects of small doses of nicotine occur in intact as well as

81
brainstem-transected animals. Nicotine acts primarily directly on
brainstem neuronal circuits to produce these effects (Domino 1967).
However, stimulation of peripheral afferents (Ginzel 1987) and
release of catecholamines and possibly neurotransmitters and modu-
lators, such as serotonin or histamine, may enhance the direct
central effects of nicotine.

The EEG-activating effects of nicotine result in behavioral arousal
(Domino, Dren, Yamamoto 1967). In cigarette smokers, nicotine
produces sedative and stimulant effects (Kumar and Lader 198)).
Aceto and Martin (1982) have reviewed the large variety of nicotine
effects on behavior including facilitation of memory, the increase in
spontaneous motor activity, nicotine’s antinociceptive properties,
and its suppression of Irritability. These behavioral and psychologi-
cal effects are discussed in Chapters IV and VI.

Distribution and Cerebral Metabolic Effects of Nicotine

Nicotine, administered by various routes, rapidly enters the brain
and also distributes to specific, peripheral organs. Nicotine produces
a distinct pattern of stimulation of cerebral metabolic activity that
suggests where nicotine acts in the brain. This Section reviews
studies on the distribution of nicotine after its administration to
experimental animals, data on the relationship between tissue levels
of nicotine and the drug’s biological effects, and studies on mapping
the cerebral metabolic effects of nicotine in the rat brain.

Distribution of Nicotine

Tissue Distribution of Nicotine: Time Course and Other
Considerations

The distribution in the body of exogenously administered nicotine
has been a topic of interest for more than a century and has been
reviewed several times (Larson, Haag, Silvette 1961; Larson and
Silvette 1968, 1971). As early as 1851, Orfila described experiments
in which he detected nicotine in various organs (e.g., liver, kidney,
lungs) and in the blood of animals after nicotine administration. In
the 1950s the development of radiotracer methods led to a reexami-
nation of nicotine distribution in the body.

Werle and Meyer (1950} found that the brain, compared with other
organs, contained the highest nicotine levels immediately after
injection of a lethal dose in guinea pigs. Tsujimoto and colleagues
(1955) found a high concentration of nicotine in the brain after the
drug was administered to rabbits and dogs. Yamamoto (1955)
observed that 1 hr after a subcutaneous (s.c.) injection of 5 mg/kg in
the rabbit, the nicotine content was highest in the kidney. The
pancreas, ileum, ventricular muscle, skeletal muscle, lung, spleen,
cerebral cortex, omental fat, and liver showed progressively lower

82
levels of nicotine at 1 hr. None of the tissues had detectable levels at
6 hr. In the dog, the highest level at 1 hr was in the kidney, followed
by the pancreas, brain, ileum, liver and omental fat, spleen, heart,
muscle, and lung.

Schmiterléw and colleagues used radiolabeled nicotine and whole-
body autoradiography to study the distribution of nicotine in several
species (Hansson and Schmiterléw 1962; Appelgren, Hansson,
Schmiterlow 1962, 1963; Hansson, Hoffman, Schmiterlow 1964;
Schmiterloéw et al. 1965; Schmiterlow et al. 1967). After radiolabeled
nicotine was administered, radioactivity representing nicotine and
its metabolites was concentrated in some organs, particularly the
brain. Hansson and Schmiterléw (1962) injected (S)-nicotine-methy]-
'4C intramuscularly or intravenously (i.v.) in mice. Within 5 min,
high concentrations were found in the brain, adrenal medulla,
stomach wall, and kidney. Lower concentrations were observed in
the liver, skeletal muscle, and blood, but all concentrations were
higher in tissue than in blood. Activity was high in the kidney from 5
min to 4 hr after the nicotine injection, with the highest activity
occurring within the first hour. The adrenal medulla maintained a
high concentration at 1 hr and 4 hr after injection, but little or no
activity was observed at 24 hr. At 30 min, the levels were high in the
walls of large blood vessels and in the bone marrow. Radioactivity
disappeared rapidly from the brain.

Appelgren, Hansson, and Schmiterléw (1962) prepared whole-body
autoradiograms of mice and cats given i.v. injections of '*C-nicotine.
An initial, heterogeneous accumulation of radioactivity occurred in
the CNS. Fifteen minutes after the radiotracer injection, the cat
brain showed distinctly more intense labeling of grey than of white
matter. Also apparent was a regional distribution within grey
matter areas, particularly in the hippocampus. By 30 min, radioac-
tivity was reduced. Studies of mice demonstrated a high concentra-
tion of label in the brain at 5 min. By 30 min, the concentration was
high in salivary glands, stomach contents, liver, and kidneys, while
the brain was almost devoid of radioactivity. The same group also
showed the accumulation of '4C-nicotine in the retina of the eye after
iv. administration (Schmiterlow et al. 1965).

Fishman (1963) reported that in rats given randomly labeled '+C-
nicotine intraperitoneally (i.p.) and killed 3 hr later, the kidney
contained the highest concentration of radioactivity, followed by the
lung, liver, brain, skeletal muscle, spleen, and heart. In the dog,
more '4C-nicotine was present in the stomach wall than in any other
tissue analyzed 3 hr after i.v. injection of radioactive nicotine.

Yamamoto, Inoki, and Iwatsubo (1967) gave mice s.c. injections of 5
mg/kg methyl-!*C-nicotine. Five minutes later, they found 0.5 to 1
ug/g (wet weight) of nicotine in various brain regions, including the
cerebral cortex, superior and inferior portions of the brain stem, and

83
the cerebellum. Highest levels were detected 5 to 10 min after
injection. Maximum levels in liver and whole blood were observed 2
and 10 min, respectively, after the injection.

Yamamoto, Inoki, and Iwatsubo (1968) studied penetration of !4C-
nicotine in rat tissues in vivo and in vitro. They found that 5 mg/kg,
i.p., in male Wistar rats produced the following maximum tissue-to-
blood ratios of !4C-nicotine activity after 10 to 20 min: kidney, 8.7;
liver, 6.7; submaxillary gland, 6.2; cerebral cortex, 3.5; brainstem,
2.4; and heart, 1.8. When they incubated tissue slices with 10-* M 4C.
nicotine for 30 min at 37°C, the relative uptake of the label was
similar: kidney cortex, 2.6; liver, 2.1; submaxillary gland, 2.1; and
cerebral cortex, 2.0. Penetration in slices was unaffected by uncou-
pling oxidative phosphorylation or blocking metabolic pathways,
indicating that the uptake was not by active transport. In vivo,
tissue-to-blood ratios were greater than slice-to-medium ratios,
indicating that a process other than passive diffusion was involved.

Because the respiratory tract is a major route by which nicotine
from tobacco smoke enters the body, Schmiterléw and coworkers
(1965) sprayed !*C-nicotine solution directly onto the trachea of mice.
Autoradiograms from mice killed at 2 min exhibited a high amount
of radioactivity in the respiratory tract and lungs and showed that
nicotine enters the CNS rapidly by this route as well. At 15 min,
radioactivity still persisted in the lungs, was reduced in the brain,
and appeared in large amounts in the kidneys and stomach.

Uptake and distribution of nicotine from tobacco smoke have also
been assessed. Harris and Negroni (1965) exposed mice to cigarette
smoke and extracted nicotine from the lungs (5 to 25 ug). Mattila and
Airaksinen (1966) exposed guinea pigs to the smoke of one 4-g cigar
over a period of 40 min, with intermittent ventilation with fresh air,
and found that the same tissues which concentrated nicotine
administered by other routes also showed nicotine uptake from
smoke. Organ-to-blood ratios were lung, 2.0; spleen, 3.0; intestine,
2.9; and brain, 1.1.

The use of positron-emitting radiotracers permits in vivo estima-
tion of nicotine uptake into the brain and other organs, offering the
potential of eventually relating nicotine action in the living human
brain to behavioral and disease states. Maziere and coworkers (1976)
prepared (S)-nicotine-methyl-''C, which they administered by i.v.
injection to mice and rabbits. The time course of the radiotracer
confirmed earlier studies and showed a maximum concentration in
the 5 min following injection, except in the liver and spleen. Highest
radioactivity was in kidneys and brain, followed by liver and lungs.
The brain activity dropped rapidly, whereas the kidney concentra-
tion remained high (8 percent of injected dose) at 50 min after the
injection. External imaging by a y camera showed considerable

84
radioactivity in the head, kidneys, and liver. Brain activity decreased
sharply over 1 hr, while activity remained high in liver and kidneys.

Maziere and coworkers (1979) used ''C-nicotine and positron
emission tomography (PET) in baboons and found that *?C-nicotine
readily penetrated into the brain and then dropped sharply with
time. Radioactivity was high in the temporal lobe, cerebellum,
occipital cortex, pons, and medulla oblongata. There was also a high,
stable radioactivity level in the retina, consistent with the earlier
observation that radioactivity from '*C-nicotine is found in the
retina after iv. administration (Schmiterlow et al. 1965).

Heterogeneity of Nicotine Uptake: Microautoradiographic and
Subcellular Studies

Appelgren, Hansson, and Schmiterlow (1963) used a microautora-
diographic method to study the localization of nicotine within the
superior cervical ganglion of the cat. Most of the radioactivity was
localized in the ganglion cells, with little labeling of satellite cells
and connective tissue.

Schmiterlow and coworkers (1967), using microautoradiograms of
mouse brains after injection of '*C-nicotine and *H-nicotine, reported
that nicotine is concentrated in nerve cells. Brain areas with a high
density of nerve cells, such as the molecular and pyramidal cell
layers of the hippocampus and the molecular layer of the cerebel-
lum, contained high amounts of radioactivity.

Yamamoto, Inoki, and Iwatsubo (1967) studied accumulation of
14C-nicotine into subcellular fractions (nuclear, mitochondrial, nerve
ending, microsomal, soluble) of mouse brain after i.p. injection of 5
mg/kg (20 wCi/kg). Most of the radioactivity was in the soluble
fraction. Less than one-tenth of the radioactivity in the soluble
fraction was found in microsomes and nerve endings; however,
radioactivity levels in microsomes were somewhat higher than in
nerve endings.

Effects of Nicotine on Cerebral Metabolism

Following the demonstration that *H-nicotine binds stereoselec-
tively and specifically in preparations of rat brain (Yoshida and
Imura 1979; Martin and Aceto 1981; Marks and Collins 1982), brain
binding sites were visualized (Clarke, Pert, Pert 1984) and quantified
(London, Waller, Wamsley 1985) by light microscopic autoradiogra-
phy. However, mapping nicotinic binding sites or identifying specific
binding sites for any drug or neurotransmitter does not necessarily
mean that receptors are coupled to pharmacologic actions. An
example of nonfunctional, stereoselective, specific binding is that of
*H-naloxone to glass fiber filters (Hoffman, Altschuler, Fex 1981). In
addition, because the brain is a highly interconnected organ, drugs

85
may produce effects in brain regions remote from their initial
receptor interactions. Receptor maps would show primary binding
sites but not sites where important secondary actions might occur.

Functional mapping procedures, such as the use of autoradio-
graphic techniques to measure rates of LCGU and regional cerebral
blood flow, are another way to determine the sites of the in vivo
effects of nicotine in the brain. The 2-deoxy-D-[1-'*C]glucose (2-DG)
method for measuring LCGU (Sokoloff et al. 1977) has been used to
demonstrate a relationship between local cerebral function and
glucose utilization under a wide variety of experimental conditions,
including pharmacologic treatments (Sokoloff 1981; McCulloch
1982). The effects of acute, s.c. injections of nicotine on LCGU were
examined by London and colleagues (1985, 1986) and by London,
Szikszay, and Dam (1986), while Griinwald, Schrock, and Kuschinsky
(1987) measured the effects on LCGU of constant plasma levels of
nicotine produced by i.v. infusion.

Subcutaneous injections of nicotine stimulated LCGU in specific
brain regions (Table 1, Figure 1), including portions of the visual,
limbic, and motor systems. Effects of nicotine infusion generally
paralleled those obtained with s.c. injections. The greatest increase
in response to s.c. nicotine occurred in the medial habenula. Marked
increases in LCGU were noted in the anteroventral thalamic
nucleus, interpeduncular nucleus, and superior colliculus. Moderate
increases were seen in the retrosplenial cortex, interanteromedial
thalamic nucleus, lateral geniculate body, and ventral tegmental
area. No significant effects were observed in the frontoparietal
cortex, lateral habenula, or central grey matter. LCGU responses to
s.c. injection of nicotine were completely blocked by mecamylamine,
indicating the specificity of nicotine effects.

The effects of nicotine on LCGU correlate well with the distribu-
tions of *H-nicotine binding sites (Clarke, Pert, Pert 1984; London,
Wailer, Wamsley 1985). Areas such as the thalamic nuclei, the
interpeduncular nucleus, medial habenula, and the superior collicu-
lus, where there is dense labeling with *H-nicotine, show moderate to
marked nicotine-induced LCGU increases. Areas with less specific
binding show smaller LCGU responses to nicotine, and the central
grey matter, which lacks specific *H-nicotine binding, shows no
LCGU response. Similarly, nicotine dramatically increases LCGU in
the medial but not the lateral habenula, reflecting different densities
of °H-nicotine binding sites. In general, *H-nicotine binding sites
visualized autoradiographically in the rat brain are functional
nicotine receptors. However, layer IV of the neocortex displays
significant *H-nicotine binding, but lacks an LCGU response.

In most brain areas, significant LCGU stimulation was obtained
with 0.3 mg/kg of nicotine s.c. (London et al. 1986), a dose similar to
one used successfully in training rats to distinguish nicotine from

86
TABLE 1.—R,S-Nicotine effects on glucose utilization in the

 

 

 

rat brain
Local cerebral glucose utilization
(umol/100 g tissue/minute)
Brain region Saline control Nicotine (1.75 mg/kg)
Frontoparietal cortex, layer [IV 110 + 81 108 + 6.5
Retrosplenial cortex. layer I 98 + 6.5 123 + 5.1?

Thalamic nuclei

Anteroventral 109 + 6.5 201 + 6.17

Interanteromedia! 125 + 86 175 + 12.3'
Lateral geniculate body 82 = 68 106 + 44!
Interpeduncular nucleus 99 + 98 182 + 9.3!
Medial habenula 70 + 52 167 + 3.7)
Superior colliculus 72 + 5.2 142 = 46!
Central grey matter 66 + 4.0 V7 + 43

 

NOTE: Results are expressed as the means plus or minus standard deviation for four rats per group.
‘Significantly different from saline contro! (p< 0.05).
SOURCE: London et al. (1985).

 

FIGURE 1.—Effect of subcutaneous R,S-nicotine (1 mg/kg, 2
min before 2-deoxyglucose) on
autoradiographic grain densities, representing
glucose utilization

NOTE: Photographs of x-ray film exposed to 20-;:m brain sections from controi rat (A) given 0.9 percent sodium
chloride 11 mL/kg! and another rat (B) given nicotine; note the increased density in medial habenula (mh) and
fasciculus retroflexus (fr

SOURCE: London et al. (1986).

saline in a T-maze apparatus (0.4 mg/kg, s.c.) (Overton 1969).
Nicotine-induced stimulation of LCGU in the ventral tegmental area

87
and the habenular complex (London et al. 1985, 1986) may relate to
the reinforcing properties of the drug (see Chapter IV). These regions
of the brain have been implicated in drug- and stimulation-induced
reward systems, respectively (Wise 1980; Nakajima 1984). Additional
studies, using specific conditions under which nicotine is reinforcing,
are needed to elucidate the anatomical loci involved in nicotine-
induced reward and to identify the neurophysiological mechanisms
by which nicotine acts as a reinforcer.

Nicotine Receptors

Nicotine exerts diverse pharmacologic effects in both the peripher-
al nervous system (PNS) and CNS. The peripheral actions of nicotine
are important, and some may reinforce the self-administration of
nicotine. For example, stimulation in the trachea (Rose et al. 1984)
seems to be involved in some of the pleasurable effects of smoking.
Skeletal muscle relaxation and electrocortical arousal, both stimu-
lated by actions of nicotine in the lung (Ginzel 1967a,b, 1975, 1987),
may contribute to habitual tobacco use (Chapter VI). However, it is
generally believed that the central actions of nicotine are of primary
importance in reinforcing tobacco use (Chapter IV). In animals, the
neuropsychopharmacologic effects of this drug are, with few if any
exceptions, mediated through central sites of action. These effects
are likely to contribute to the drug’s reinforcing properties in
animals and humans (Clarke 1987b). In addition, the effects of
nicotinic antagonists on tobacco smoking in humans (Stolerman et
al. 1973) and in rhesus monkeys (Glick, Jarvik, Nakamura 1970)
suggest a central site of reinforcement, but do not rule out a
peripheral site. To understand these actions, it is important to know
exactly where nicotine acts in the body. This Section discusses
evidence for nicotine receptors.

Peripheral Nicotine Receptors

In the mammalian PNS, nicotine and muscarine mimic different
actions of ACh by acting at different types of cholinergic receptors.
Nicotinic cholinergic receptors (nAChRs) have been subdivided
according to location and sensitivity to nicotinic antagonists. Recep-
tors of the C6 or “ganglionic” type are found principally at
autonomic ganglia, in the adrenal medulla, and at sensory nerve
endings; nicotinic cholinergic transmission in autonomic ganglia is
selectively blocked by hexamethonium and certain other compounds.
Receptors of the “neuromuscular” type (sometimes referred to as
C10 type) are located on the muscle endplate, where transmission is
selectively blocked by compounds such as decamethonium and alpha-
bungarotoxin (a-BTX).

88
Higher doses of nicotine are required to stimulate nAChRs in
skeletal muscle than at autonomic ganglia. Ganglionic nAChRs
appear to be more sensitive than their neuromuscular counterparts,
not only to the stimulant but also to the desensitizing actions of
nicotine (Paton and Savini 1968). Doses of nicotine obtained by
smoking cigarettes do not appear to affect the muscle endplate
directly. Therefore, if the CNS were to possess both types of nAChR,
doses of nicotine obtained by normal cigarette smoking might affect
only the C6-receptor population. Accordingly, many of the central
effects of nicotine in vivo and in vitro are reduced or blocked by
nicotinic antagonists that are C6-selective in the periphery. The
most widely used C6-selective antagonist is mecamylamine, which
passes freely into the CNS after systemic administration. Mecamyla-
mine antagonizes actions of nicotine in the brain and spinal cord, as
revealed by behavioral (Collins et al. 1986; Goldberg, Spealman,
Goldberg 1981) and electrophysiological experiments (Ueki, Koketsu,
Domino 1961) and also by studies of neurotransmitter release (Hery
et al. 1977; Chesselet 1984). There have been few attempts to
determine whether these central nicotinic actions are also blocked
by neuromuscular antagonists, while several studies support the
existence of central C6 nAChRs (Aceto, Bentley, Dembinski 1969;
Brown, Docherty, Halliwell 1983; Caulfield and Higgins 1983; Egan
and North 1986).

The search for putative central a-BTX nAChRs has been hindered
by several factors, including the central convulsant actions of a-BTX
antagonists (Cohen, Morley, Snead 1981) and the probable need to
deliver locally high concentrations of nicotine. Nevertheless, several
studies have demonstrated actions of nicotine or cholinergic agonists
that can be reduced or blocked by a-BTX, which acts selectively at
neuromuscular nAChRs (Zatz and Brownstein 1981; Farley et al.
1983; de la Garza et al. 1987a).

Radioligand Binding to Putative Nicotine Cholinergic
Receptors in Mammalian Brain

Many receptors for neurotransmitters in the brain have been
identified through the use of radiolabeled probes (radioligands).
Attempts to label putative brain nAChRs have used compounds with
known potency at peripheral sites (see Table 2).

Agonist Binding

The stereospecific, saturable, and reversible binding of *H-nicotine
to rodent brain is well-described (Romano and Goldstein 1980; Marks
and Collins 1982; Costa and Murphy 1983; Benwell and Balfour
1985a; Clarke, Pert, Pert 1984). Most studies have demonstrated the
existence of a population of high-affinity binding sites (reflected by a
dissociation constant in the low nanomolar range) that is potently

89
TABLE 2,—Radioligands for putative nicotinic cholinergic
receptors in mammals

 

 

Functional

Antagonists Bind antagonism Sites examined Agonists
1. BTX Yes Yes Muscle endplate ‘H-nicotine

Yes Yes Autonomic ganglia, spinal cord

Yes Yes Brain (certain sites only) °H-methyl-carbachol
‘T-naja toxin Yes Yes Muscle endplate °H-ACh (with excess

Yes ND! Brain muscarinic antagonist

and AChE inhibitor)

°H-dTC ND Yes Muscle, spinal cord, ganglia

Yes Yes Brain
*H-DHBE ND Yes Muscle, autonomic ganglia

Yes Yes Brain, spinal cord
Neosurugatoxin ND No Muscle endplate

ND Yes Autonomic ganglia

Yes Yes Brain (inhibits *H-nicotine)

 

 

‘ND=no data.

inhibited by nicotinic agonists including ACh. In contrast, most
nicotinic antagonists have very low affinity for this site. Binding
with similar characteristics has been reported in rat brain tissue
with *H-methyl-carbachol (Abood and Grassi 1986; Boksa and
Quirion 1987) and with *H-ACh in the presence of excess atropine to
prevent binding to muscarinic receptor sites (Schwartz, McGee,
Kellar 1982).

In the presence of atropine, tritiated nicotine and *H-ACh proba-
bly bind to the same population of high-affinity sites in rat brain.
Thus, the two radioligands share the same neuroanatomical distribu-
tion of binding (Clarke, Schwartz et al. 1985; Marks et al. 1986;
Martino-Barrows and Kellar 1987). Binding of both ligands is
inhibited with similar potency by a range of nicotinic agents, is up-
regulated by chronic nicotine treatment in vivo, is down-regulated by
chronic treatment with acetylcholinesterase inhibitors, and is dimin-
ished by disulfide reducing agents in vitro (Marks et al. 1986;
Martino-Barrows and Kellar 1987; Schwartz and Kellar 1983).
Although less well studied, it appears that sites labeled by *H-
methyl-carbachol are the same as those labeled by 3H-ACh and °H-
nicotine (Abood and Grassi 1986; Boksa and Quirion 1987). High-
affinity nicotine binding sites have been found in brain tissue of mice
(Marks and Collins 1982), rats (Romano and Goldstein 1980),
monkeys (Friedman et al. 1985), and humans (Shimohama et al.
1985; Flynn and Mash 1986; Whitehouse et al. 1986).

Some investigators have reported a second class of sites which are
characterized by lower binding affinity and higher capacity for *H-

90
nicotine. With no demonstrated differential anatomical distribution
or stereoselectivity (Romano and Goldstein 1980; Marks and Collins
1982; Benwell and Balfour 1985b), these low-affinity sites are of
questionable pharmacologic significance, but may be the result of
post mortem proteolysis (Lippiello and Fernandes 1986). Careful
analysis of *H-nicotine binding conducted in the absence of protease
inhibitors has revealed the existence of five affinity sites or states
(Sloan, Todd, Martin 1984). Functional studies (Martin et al. 1986)
suggest that some of these different sites may represent in vivo sites
of action for nicotine, although it is not clear which if any would be
activated by nicotine doses obtained from typical cigarette smoking.

Radioligand Binding

Many receptors of different nicotine binding affinities have been
reported. It is unclear whether these reflect different conformational
states or binding sites of a single type of receptor, distinct receptor
populations, or a single type of high-affinity site which has under-
gone proteolytic degradation. Preliminary evidence supports the
existence of distinct receptor subtypes labeled by agonists. Two
components of high-affinity *H-nicctine binding, differing in their
affinity for neosurugatoxin, can be distinguished in rat brain. The
relative proportion of these two components differs in different
regions of the rat brain, suggesting that they are physically distinct
receptors (Yamada et al. 1985).

Antagonist Binding

Most studies of nicotine binding in mammalian brain have used
radioiodinated a-BTX (*7°I-BTX), which binds with high affinity and
in a saturable manner to sites in mammalian brain (Schmidt, Hunt,
Polz-Tejera 1980; Oswald and Freeman 1981). This binding is
selectively inhibited by nicotinic agents, including nicotine and ACh.
Cobra (naja) alpha-toxin, like a-BTX, is a selective neuromuscular
blocker in the mammal, and appears to label the same sites as a-BTX
in mammalian brain. Binding is potently inhibited by unlabeled a-
BTX and has a regional distribution resembling that of '*°I-BTX
binding (Speth et al. 1977). The antagonist dihydro-beta-erythroidine
(DHBE) binds to two sites in rat brain, but the regional distribution
of binding differs from that of '?°I-BTX (Williams and Robinson
1984). DHBE acts with similar potency at both types of peripheral
nAChR in vivo. It is not clear whether “H-d-tubocurarine binding is
selectively inhibited by nicotinic agents. In rat brain, '*°I-BTX binds
to a distinct population of sites that are not labeled with high affinity
(nanomolar kD) by tritiated nicotinic agonists. Radioiodinated a-
BTX sites have a different neuroanatomical distribution (Marks and
Collins 1982; Schwartz, McGee, Kellar 1982; Clarke, Schwartz et al.

91
1985) and can be physically separated from tritiated agonist binding
sites by affinity chromatography (Schneider and Betz 1985; Wonna-
cott 1986). This type of study helps to determine the location and
numbers of nicotine binding sites.

Functional Significance of Nicotinic Binding Sites
High-Affinity Agonist Binding Sites

Brain sites which bind *H-ACh and *H-nicotine with high affinity
represent nAChRs that respond in some ways like the C6 type of
receptor found in the periphery (Clarke 1987a). Studies using the 2-
DG technique have revealed that the neuroanatomical pattern of
cerebral activation following the systemic administration of nicotine
in rats is strikingly similar to the distribution of high-affinity agonist
binding demonstrated autoradiographically (London et al. 1985;
Grunwald, Schrok, Kuschinsky 1987). Pretreatment with mecamyla-
mine blocks the effects of nicotine on LCGU, suggesting that
putative ganglionic (C6-type) receptors in the brain are associated
with high-affinity agonist binding.

Most of nicotine’s actions on central receptors are blocked by the
C6-selective antagonist mecamylamine. The relevant nAChRs are
probably those which are labeled with high affinity by tritiated
agonists. However, the absence of high-affinity agonist binding sites
in PC12 cells (derived from a pheochromocytoma cell line) known to
express C6-type receptors (Kemp and Morley 1986) indicates that
although central and ganglionic nAChRs have pharmacologic simi-
larities, they may not be identical at the molecular level.

High-affinity agonist binding sites are relevant to long-term effects
of human tobacco smoking. Recently, Benwell, Balfour, and Ander-
son (in press) observed that the density of high-affinity *H-nicotine
binding in post mortem human brain is higher in smokers than in
nonsmokers. The increased density of sites in smokers is consistent
with studies in animals that show that chronic treatment with
nicotine leads to an increased number of nicotinic receptors in the
brain (Schwartz and Kellar 1983; Marks, Burch, Collins 1983b).

Alpha-Bungarotoxin Binding Sites

Although a-BTX does not block nicotinic actions in all areas of the
CNS (Duggan, Hall, Lee 1976; Egan and North 1986), there are
several reports of antagonism (Zatz and Brownstein 1981; Farley et
al. 1983; de la Garza et al. 1987a). In the rat cerebellum, locally
applied nicotine alters single-unit activity in a manner dependent on
cell type: nicotine excites interneurons but inhibits Purkinje cells.
Both actions are directly postsynaptic (de la Garza et al. 1987, in
press(b)). The inhibitory effects of nicotine are blocked by hexame-

92
thonium but not by a-BTX, which does block the excitatory effects
(de la Garza et al., in press(a)).

Strain differences exist in mice in the physiological and behavioral
effects of nicotine, in the development of tolerance to these effects,
and in the regional distribution of /#5]-BTX binding density (Marks,
Burch, Collins 1983a; Marks, Stitzel, Collins 1986). The genetically
determined variation in response is not readily explained by
differences in brain nicotinic receptors. However, a classical genetic
analysis indicates that the density of !*°I-BTX binding sites in mouse
hippocampus correlates with susceptibility to seizures induced by
high doses of nicotine (Miner, Marks, Collins 1984). These and other
considerations (Clarke 1987a) suggest that 1*5I]-BTX may label a
subtype of nAChR in the brain and that this receptor is pharmaco-
logically akin to the nAChR found in muscle.

Although '*5I-BTX binding sites are found in human brain, the
available evidence suggests that nicotine at doses obtained from
cigarette smoking does not activate this population of brain nAChRs.
Rather, the pattern of neuronal activation that follows the in vivo
administration of nicotine in animal experiments, even in doses far
greater than those likely to occur during smoking, resembles the
neuroanatomical distribution of high-affinity agonist binding sites
(London et al. 1985; Grunwald, Schrok, Kuschinsky 1987). However,
this issue is not conclusively resolved, and a potential role for
bungarotoxin binding receptors in mediating effects of smoking
cannot be completely excluded.

Behavioral and Physiological Studies

The effects of mecamylamine on several responses elicited by
nicotine in mice have been examined (Collins et al. 1986). The
responses are of two major classes: those blocked by low doses of
mecamylamine (inhibitory concentrations for 50 percent of mice
tested (IC;,) <0.1 mg/kg) (seizures and startle response) and those
blocked by higher doses (IC;, approximately 1 mg/kg) (effects on
respiratory, heart rate, body temperature, and Y-maze activity).
Strain differences are also apparent in the sensitivity to mecamyla-
mine blockade. These findings are consistent with the existence of at.
least two types of central nAChR.

The Neuroanatomical Distribution of Nicotinic Binding
Sites in the Brain

High-Affinity Agonist Binding Sites
Rodent

Autoradiographic maps of high-affinity nicotinic binding sites in
rat brain are essentially identical for *H-nicotine, 9>H-ACh, and ?H-
methy!-carbachol (Clarke, Pert, Pert 1984; Clarke, Schwartz et al.

93
1985; London, Waller, Wamsley 1985; Boksa and Quirion 1987).
Dense labeling is observed (1) in the medial habenula and interpe-
duncular nucleus, which appear to belong to a common cholinergic
system; (2) in the so-called specific motor and sensory nuclei of the
thalamus and in layers ITI and IV of cerebral cortex with which they
communicate; (3) in the substantia nigra pars compacta and ventral
tegmental area, where labeling is associated with dopaminergic cell
bodies (Clarke and Pert 1985); and (4) in the molecular layer of the
dentate gyrus, the presubiculum, and the superficial layers of the
superior colliculus. Labeling is sparse in the hippocampus and
hypothalamus.

Monkey

The autoradiographic distribution of high-affinity *°H-nicotine
binding in rhesus monkey brain is similar to that in the rat
(Friedman et al. 1985). Dense labeling has been noted in the anterior
thalamic nuclei and in a band within cerebral cortex layer ITI. The
latter band is densest and widest in the primary sensory areas.
Several other thalamic nuclei are moderately labeled, but as in the
rat, the label is sparse in the midline thalamic nuclei. In contrast to
findings for the rat, the medial habenula appears unlabeled.

Human

High-affinity agonist binding has not been mapped autoradio-
graphically in human brain. However, assays of a few dissected brain
areas suggest the following pattern: nucleus basalis of Meynert >
thalamus > putamen > hippocampus, cerebellum, cerebral cortex,
and caudate nucleus (Shimohama et al. 1985). Two affinity sites for
°H-nicotine have been detected, and the regional distribution
observed reflects the presence of both sites.

Alpha-Bungarotoxin Binding Sites

Because ‘*°]-BTX sites may not be relevant to tobacco smoking,
they will be discussed only briefly here. There are clear differences of
regional distribution not only between mice and rats, but also
between different strains of mice (Marks et al. 1986). The autoradio-
graphic distribution of !°I-BTX labeling in rat brain is strikingly
different from the pattern of *H-agonist labeling, with highest site
density in hippocampus, hypothalamus, and superior and inferior
colliculi (Clarke, Schwartz et al. 1985). An attempt to map '*5]-BTX
binding in human brain was hampered by a high degree of
nonspecific binding, with diffuse specific labeling in the hippocam-
pus and cerebral cortex (Lang and Henke 1983).

94
Molecular Biology

Goldman and colleagues have mapped regions in the brain which
contain cell bodies expressing RNA that codes for putative nAChRs.
The RNA identified is homologous to cDNA clones encoding the
alpha subunits of the muscle nAChR and a putative neuronal
nAChR (Goldman et al. 1986; Goldman et al. 1987). These and
related findings show that a family of genes exists that codes for
proteins similar to, but not identical with, the muscle nAChR. The
functional role of these putative nAChR subtypes in the CNS is not
clear.

Central Nicotinic Cholinergic Receptors: Pre- or
Postsynaptic?

Presynaptic Regulation of Neurotransmitter Release

The release of ACh from some nerve terminals in the CNS (Rowell
and Winkler 1984; Beani et al. 1985) and periphery (Briggs and
Cooper 1982) is increased by activation of presynaptic nicotinic
“autoreceptors.” Preliminary evidence from lesion experiments
suggests that some nicotinic autoreceptors in the brain may be high-
affinity *H-nicotine binding sites (Clarke et al. 1986).

Nicotine also modulates the release of certain other neurotrans-
mitters by acting at receptors located on nerve terminals. This form
of regulation has been shown for dopaminergic, noradrenergic, and
serotonergic terminals (Starke 1977; Chesselet 1984). Lesion studies
suggest that these receptors are labeled by *H-agonists (Schwartz,
Lehmann, Kellar 1984; Clarke and Pert 1985; Prutsky, Shaw,
Cynader 1987).

Somatodendritic Postsynaptic Actions

Much of *H-agonist labeling probably represents nAChRs located
on neuronal cell bodies or dendrites. For example, nicotine excites
neurons postsynaptically in the medial habenula, locus coeruleus,
and interpeduncular nucleus, all areas of moderate to dense *H-
agonist binding (Brown, Docherty, Halliwell 1983; Egan and North
1986; McCormick and Prince 1987).

Neuroendocrine and Endocrine Effects of Nicotine

Nicotine has direct and indirect effects on several neuroendocrine
and endocrine systems (Balfour 1982; Clarke 1987a; Hall 1982). This
Section reviews research on the effects of nicotine in animals and
humans that are relevant to understanding cigarette smoking.
Nicotine effects on cholinergic and noncholinergic nicotinic recep-
tors, as well as on the release of catecholamines, monoamines,
pituitary hormones, cortisol, and other neuroendocrine chemicals,

95
are discussed. Effects on single neuroregulators are emphasized, but
it is important to recognize that there are extensive interrelation-
ships among these substances (Tuomisto and Mannisté 1985).

Nicotine has effects on peripheral endocrine as well as on central
neuroendocrine functions. In the early 1900s researchers discovered
that nicotine stimulated autonomic ganglia (ganglia were painted
with tobacco solutions), inducing such effects as the release of
adrenal catecholamines (Larson, Haag, Silvette 1961). As the health
consequences of cigarette smoking have become clearer, many
investigators have sought to determine tobacco’s effects on the
endocrine system, with the possibility that understanding such
effects may help to explain smoking behavior. Nicotine is regarded
as the major pharmacologic agent in tobacco and tobacco smoke
responsible for alterations in endocrine function. However, there has
not been a systematic evaluation of the effects of metabolites of
nicotine or constituents of tobacco other than nicotine on the
endocrine system.

The functional significance of nicotine-induced perturbations in
hormonal patterns and the role of neuroregulators in smoking are
poorly understood. Extensive literature using nicotinic agonists and
antagonists indicates relationships between cholinergic activity and
particular behavioral effects (Henningfield et al. 1983; Kumar,
Reavill, Stolerman, in press). Similar strategies have been employed
to explore the contributions of catecholamines to smoking-related
behavior. However, the exploration of the importance of neuroregu-
lators in the reinforcement of cigarette smoking is still at an early
stage.

Cholinergic Effects

Nicotine has cholinergic effects in the PNS and CNS. Nicotine is a
cholinergic agonist at peripheral autonomic ganglia and somatic
neuromuscular junctions at low doses and becomes an antagonist at
high doses (Volle and Koelle 1975). Nicotine also releases ACh in the
cerebral cortex (Armitage, Hall, Morrison 1968; Rowell and Winkler
1984) and in the myenteric plexus of the peripheral ANS (Briggs and
Cooper 1982). Balfour (1982) has suggested that cortical arousal (see
Electrophysiological Actions of Nicotine for a detailed discussion) is
mediated by ACh release but that behavioral stimulation (see
Chapter IV) either is not mediated by ACh release or does not
depend on the action of ACh at a muscarinic receptor.

Studies involving intracerebral administration of nicotine have
been used to determine the loci of nicotine’s action (Kammerling et
al. 1982; Wu and Martin 1983). The injection of nicotine into the
cerebral ventricles of cats, dogs, and rats produces a variety of effects
including changes in cardiovascular activity, body temperature,
respiration, salivation, muscle reflex tone, and electrocortical indices

96
of sleep and arousal; the direction and duration of effects depend on
dosage and on baseline response parameters (Hall 1982).

Nicotine’s cholinergic actions can affect other neuroregulators in
the body (Andersson 1985). Nicotine stimulates NE release in the
hypothalamus by a Ca*--dependent process that can be inhibited by
prior administration of hexamethonium or ACh (Hall and Turner
1972; Westfall 1974). The mechanism resembles nicotine’s effects on
peripheral adrenergic nerve terminals (Westfall and Brasted 1972),
At high dose levels, nicotine stimulates NE release by displacing it
from vesicle stores at sites outside the hypothalamus (Balfour 1982).
These actions are relevant to understanding the reinforcing effects
of nicotine. For example, using drug discrimination procedures,
Rosecrans (1987) has demonstrated that intact central NE and
dopamine (DA) function were required to elicit the cue properties of
nicotine.

Intravenous administration of nicotine modulates the release of
both neurohypophyseal and adenohypophyseal hormones (Bisset et
al. 1975; Hall, Francis, Morrison 1978). Hillhouse, Burden, and Jones
(1975) found that the in vitro application of ACh to the hypophysio-
tropic area of the rat caused a significant increase in the basal
secretion of corticotropin-releasing hormone (as measured by bioas-
say), which in turn controls, via the anterior pituitary, the release of
the pro-opiomelanocortin (POMC) group of hormones—f-endorphin,
B-lipotropin, melanocyte-stimulating hormone-releasing factor, and
adrenocorticotropic hormone (ACTH) (Meites and Sonntag 1981).
The humoral mechanism for the release of vasopressin has been
traced from the medulla to the paraventricular nuclei of the
hypothalamus (Bisset et al. 1975; Castro de Souza and Rocha e Silva
1977). Similarly, Risch and colleagues (1980) have demonstrated a
cholinergic mechanism for the release of B-endorphin.

Modulation of Catecholamine and Serotonin Activity

Dale and Laidlaw (1912) found that the pressor response of the cat
to nicotine was due in part to the release of epinephrine from the
adrenal glands. Over the past 75 years, a large body of research has
confirmed and further investigated this phenomenon. Stewart and
Rogoff (1919) quantified the effect of nicotine on adrenal epinephrine
release. Kottegoda (1953) observed that nicotine releases catechol-
amines from extra-adrenal chromaffin tissues. Watts (1961) demon-
strated the effect of smoking on adrenal secretion of epinephrine.
Hill and Wynder (1974) reported that increasing the nicotine content
in cigarette smoke progressively increased the serum concentration
of epinephrine, but not NE. Winternitz and Quillen (1977) found that
the excretion of urinary catecholamines tended to be higher on
smoking days than on nonsmoking days. Several recent studies have
focused on the role of nicotine and the mechanisms involved in the

97
release of catecholamines from cultured chromaffin cells (Forsberg,
Rojas, Pollard 1986). Earlier experiments by Douglas and Rubin
(1961), using denervated perfused cat adrenal glands, indicated that
nicotine augments catecholamine release from chromaffin cells by
promoting an influx of extracellular calcium. Forsberg, Rojas, and
Pollard (1986) suggested that nicotine-induced catecholamine secre-
tion may be mediated by phosphoinositide metabolism in bovine
adrenal chromaffin cells.

The anatomical localization and importance of biogenic mono-
amines such as serotonin (5-HT [5-hydroxytryptamine]), DA, and NE
have been the subject of intense research for the past 30 years. The
classic studies of Dahlstrom and Fuxe (1966) revealed that neurons
containing these amines were localized in specific ascending projec-
tion systems; descending monoaminergic neurons have also been
described. The physiological integrity of these systems was further
demonstrated by Aghajanian, Rosecrans, and Sheard (1967), who
observed that stimulation of 5-HT cell bodies localized in the
midbrain raphe nucleus released 5-HT from nerve endings located in
the more rostral forebrain. The recognition that these amine systems
constitute a unique interneuronal communication system has played
a central role in understanding underlying neurochemical and
behavioral mechanisms.

The cholinergic system has undergone a similar analysis (Fibiger
1982), but the delineation of specific cholinergic pathways has been
more difficult because no histochemical method has been available
for ACh. It does appear, however, that the cholinergic system is
similarly organized and interacts with specific biogenic amine
pathways. For example, Robinson (1983) has clearly shown that both
5-HT and DA systems exert tonic inhibitory control over ACh
turnover in both the hippocampus and frontal cortex regions.
Lesions of the medial raphe nuclei increase the ACh turnover rate in
hippocampal sites, while lesions of the dorsal raphe elicit a similar
effect in frontal cortical areas. Evidence of DA control comes from
the observation that the catecholamine neurotoxin, 6-OHDA, when
injected into the DA-rich septal area, facilitated hippocampal ACh
turnover. The research of Kellar, Schwartz, and Martino (1987) and
others also suggests that nicotinic receptors may occupy a presynap-
tic site on select DA and 5-HT nerve endings. Westfall, Grant, and
Perry (1983), using a tissue slice preparation, have shown that the
DMPP-induced stimulation of nicotinic receptors in the striatum will
facilitate the release of both 5-HT and DA. This preparation is devoid
of cell bodies or 5-HT- and DA-containing axon terminals, suggesting
that these nicotinic cholinergic receptors are primarily presynaptic.
Further, hexamethonium, but not atropine, attenuated nicotine-
induced amine release, confirming that these effects are nicotinic in
nature.

98
Nicotine may have simultaneous actions on many types of
neurons. Even though only one kind of receptor may be stimulated,
either activation or inhibition of a particular 5-HT, NE, or DA
neuron may be the ultimate outcome. Conversely, the activity of
specific cholinergic neurons may also be controlled by one of these
biogenic-amine-containing projection systems. Nicotine appears to
produce its discriminative stimulus effect in at least one major brain
area, the hippocampus. This site is rendered insensitive if DA
neurons innervating this area are destroyed (Rosecrans 1987). The
interrelationships of these amine pathways are important to under-
stand nicotine’s effects on behavior and its effects on the neuroendoc-
rine system because of the central role that these amine systems
play in the hypothalamic control of the pituitary.

Effects on Serotonergic Neurons

Research evaluating the relationship between nicotine and 5-HT
has involved several different approaches. Hendry and Rosecrans
(1982) compared the effects of nicotine on conditioned and uncondi-
tioned behaviors in rats selected for differences in physical activity
and 5-HT turnover. Balfour, Khuller, and Longden (1975) observed
that acute doses of nicotine were capable of attenuating hippocampal
5-HT turnover, an effect specific to the hippocampus. Fuxe and
colleagues (1987) did not observe any acute changes in 5-HT function
following acute nicotine dosing but did observe a significant reduc-
tion of 5-HT turnover following repeated doses (3 x 2 mg/kg/hr). This
effect, however, was suggested to be due to cotinine, the primary
metabolite of nicotine.

In addition to attempts to correlate 5-HT function with some
pharmacologic effect of nicotine, investigators have evaluated poten-
tial links between 5-HT and neuroendocrine function. Balfour,
Khuller, and Longden (1975) showed a relationship between 5-HT
and nicotine’s ability to induce the release of plasma corticosterone,
presumably by activation of the pituitary-adrenal axis. Following
acute nicotine injections in the rat, a reduction in 5-HT turnover
correlated with an increase in plasma corticosterone. Rats exhibited
tolerance to pituitary activation following repeated nicotine doses,
but not to the attenuation of hippocampal 5-HT turnover. Stress
antagonized nicotine-induced reductions of hippocampal 5-HT. Also,
nicotine was reported to inhibit the adaptive response to adrenocorti-
cal stimulation following chronic stress (Balfour, Graham, Vale
1986). One interpretation of these data is that nicotine can modify
how rats adapt to stress, which may be mediated by changes in
hippocampal 5-HT function. At this point, however, it is difficult to
draw firm conclusions concerning how nicotine affects 5-HT neurons
and whether this neurotransmitter is involved in any of nicotine’s

99
effects on neuroendocrine function. Hippocampal 5-HT turnover
appears to be selectively attenuated by nicotine.

Effects on Catecholaminergic Neurons

Studies of the effects of nicotine on NE-containing neurons have
produced mixed results. Earlier work suggested that nicotine may
affect behavior via a NE component, but recent research has not
supported such claims (Balfour 1982). It has been reported that
nicotine releases DA from brain tissue (Westfall, Grant, Perry 1983).
Lichtensteiger and colleagues (1982) observed that nicotine releases
DA through an acceleration of the firing rate of DA cell bodies
located in substantia nigra zona compacta when nicotine is adminis-
tered via iontophoretic application or s.c. (0.4 to 1.0 mg/kg). This
activation was marked by a significant increase in striatal DA
turnover; DHBE, but not atropine, attenuated nigrostriatal activa-
tion. Evidence that nicotine facilitates the firing of DA cell bodies by
stimulating nicotinic cholinergic receptors has recently been report-
ed by Clarke, Hommer, and coworkers (1985), who showed a specific
effect of nicotine antagonized by mecamylamine on pars compacta
cell bodies. Connelly and Littleton (1983) noted that DA release from
synaptosomes lacked stereoselectivity but was blocked by the
ganglionic-blocking drug pempidine.

Fuxe and coworkers (1986, 1987) have studied nicotine’s effects on
central catecholamine neurons in relation to neuroendocrine func-
tion. These investigators use quantitative histofluorometric tech-
niques that measure the disappearance of catecholamine stores by
administering a tyrosine hydroxylase inhibitor (AMPT) to rats
receiving various doses of nicotine or exposed to tobacco smoke.
Tissues are then exposed to formaldehyde gas, and histofluorescence
in AMPT-treated rats is evaluated in comparison to controls.

Nicotine is a potent activator of both DA and NE neuron systems
located primarily in the median eminence and in areas of the
hypothalamus. These effects result from a stimulation of nicotinic
cholinergic receptors, generally antagonized by mecamylamine.
Intermittent nicotine dosing (4 x 2 mg/kg, s.c. every 30 min) or
tobacco smoke exposure (rats were exposed to one to four cigarettes
with a smoking machine-determined nicotine yield of 2.6 mg; rats
received 8 puffs at 10-min intervals) results in a decrease of
prolactin, thyroid-stimulating hormone (TSH), and luteinizing hor-
mone (LH) and an increase of plasma corticosterone levels. Nicotine
doses of 0.3 mg/kg administered i.v. induce an overall activation of
the hypothalamic-pituitary axis, causing an increase of both ACTH
and prolactin that subsides within 60 min. Tolerance to the
corticosterone response develops after repeated nicotine doses, and
there is evidence that it develops after a single dose of nicotine
(Sharp and Beyer 1986; Sharp et al. 1987). Restraint stress increases

100