Dopamine R Gic

CHAPTER FOUR
Brain Neurons Partly Expressing

Dopaminergic Phenotype:
Location, Development,
Functional Significance,
and Regulation
Michael V. Ugrumov
Institute of Developmental Biology and Centre for Brain Research, Russian Academy of Sciences,
Moscow, Russia
Institute of Normal Physiology RAMS, Moscow, Russia
Contents
1. Introduction 38
2. Neurons Partly Expressing DA-ergic Phenotype in Ontogenesis 42
2.1 Specific characteristics 42
2.2 Distribution of “monoenzymatic” neurons in the brain 44
3. Neurons Partly Expressing DA-ergic Phenotype in Adulthood 51
3.1 Specific characteristics 51
3.2 Distribution of monoenzymatic neurons in the brain 52
4. Functioning of Monoenzymatic Neurons 65
4.1 Monoenzymatic neurons expressing TH 65
4.2 Monoenzymatic neurons expressing AADC 69
4.3 Monoenzymatic neurons expressing either TH or aromatic L-amino acid as
a functional unit 70
5. Conclusion 76
5.1 Functional significance of monoenzymatic neurons in norm and pathology 76
5.2 Regulation of monoenzymatic neurons 79
Conflict of Interest 83
Acknowledgments 83
References 83
Abstract
In addition to catecholaminergic neurons possessing all the enzymes of catecholamine
synthesis and the specific membrane transporters, neurons partly expressing the cate-
cholaminergic phenotype have been found a quarter of a century ago. Most of them
express individual enzymes of dopamine (DA) synthesis, tyrosine hydroxylase (TH), or
Advances in Pharmacology, Volume 68 # 2013 Elsevier Inc. 37

ISSN 1054-3589 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-411512-5.00004-X
38 Michael V. Ugrumov
aromatic L-amino acid decarboxylase (AADC), lacking the DA membrane transporter

and the vesicular monoamine transporter, type 2. These so-called monoenzymatic neu-
rons are widely distributed throughout the brain in ontogenesis and adulthood being in
some brain regions even more numerous than dopaminergic (DA-ergic) neurons. Indi-
vidual enzymes of DA synthesis are expressed in these neurons continuously or tran-
siently in norm and pathology. It has been proven that monoenzymatic TH neurons
and AADC neurons are capable of producing DA in cooperation. It means that L-3,4-
dihydroxyphenylalanine (L-DOPA) synthesized from L-tyrosine in monoenzymatic TH
neurons is transported to monoenzymatic AADC neurons for DA synthesis. Such coop-
erative synthesis of DA is considered as a compensatory reaction under a failure of
DA-ergic neurons, for example, in neurodegenerative diseases like hyperprolactinemia
and Parkinson's disease. Moreover, L-DOPA, produced in monoenzymatic TH neurons, is
assumed to play a role of a neurotransmitter or neuromodulator affecting the target
neurons via catecholamine receptors.
Thus, numerous widespread neurons expressing individual complementary
enzymes of DA synthesis serve to produce DA in cooperation that is a compensatory
reaction at failure of DA-ergic neurons.
ABBREVIATIONS
AADC aromatic L-amino acid decarboxylase
AN arcuate nucleus
DA dopamine
DA-ergic dopaminergic
DAT dopamine membrane transporter
GTPC guanosine triphosphate cyclohydrolase I
L-DOPA L-3,4-dihydroxyphenylalanine
MBH mediobasal hypothalamus
ME median eminence
SCN suprachiasmatic nucleus
TH tyrosine hydroxylase
VMAT2 vesicular monoamine transporter, type 2
1. INTRODUCTION
Interneuronal signalization with classical neurotransmitters, neuro-
peptides, and neurohormones is crucial for the brain functioning and plas-
ticity. Among classical neurotransmitters, catecholamines play an important
role in neural regulations. Indeed, catecholaminergic neurons, especially
dopaminergic (DA-ergic) neurons, are widely distributed throughout
the brain controlling major brain and peripheral functions (Björklund
& Lindvall, 1984; Deutch & Roth, 2003; Girault & Greengard, 2004;
Neurons Partly Expressing Dopaminergic Phenotype 39
Hökfelt, Johansson, & Goldstein, 1984; Jaeger & Teitelman, 1992;

Palkovits & Brownstein, 1989).
Dopamine (DA) is produced in cytosol of DA-ergic neurons from
L-tyrosine by its enzymatic conversion first in L-3,4-dihydroxyphenylalanine
(L-DOPA) with tyrosine hydroxylase (TH), a rate-limiting enzyme of cate-
cholamine synthesis, and then in DA with aromatic L-amino acid decarbox-
ylase (AADC) (Fig. 4.1). It is noteworthy that the enzymatic activity of the
latter is much higher than that of the former (Moore, Riegle, & Demarest,
1985). DA is captured from cytosol into the secretory granules (dense-cored
vesicles) with the vesicular membrane transporter, type 2 (VMAT2), and then
released into the extracellular space by exocytosis (Hoffman, Hansson,
Mezey, & Palkovits, 1998; Weihe, Depboylu, Schütz, Schäfer, & Eiden,
2006). After the neurotransmitter action on the target neurons and subsequent
partial enzymatic degradation, DA is captured with the DA membrane trans-
porter (DAT) from the extracellular space into the DA-ergic neurons for
reutilization (Fig. 4.2) (Hoffman et al., 1998; Ugrumov, 2008).
COOH
CH2 C NH2
H
HO L-Tyrosine
Tyrosine hydroxylase
COOH
CH2 C NH2
HO H
L-DOPA
HO
Aromatic L-amino acid

decarboxylase
HO CH2 CH2 NH2
Dopamine
HO
Figure 4.1 Dopamine (DA) synthesis from L-tyrosine. L-DOPA, L-3,4-

dihydroxyphenylalanine.
DNA • •• •
••••
••
•• R
Transcription ••• ••
pre-mRNA VMT •• •
•
Splicing •••
mRNA T D
A X DA •
•
•
T D •
A X DA •
R
• • •
• • •
• •
Synthesis of DAT• •
enzymes (D, T) TC
Figure 4.2 Schematic representation of DA turnover in dopaminergic (DA-ergic) neu-

rons. A, L-tyrosine; D, aromatic L-amino acid decarboxylase; DAT, dopamine membrane
transporter; DA, dopamine; R, dopamine receptor; T, tyrosine hydroxylase; TC, target
cell; VMT, vesicular monoamine transporter, type 2; X, L-3,4-dihydroxyphenylalanine;
points, DA molecules. Modified from Ugrumov (2008).
Figure 4.3 Schematic representation of location of major clusters of DA-ergic neurons

(dark triangles) in the brain (А8–А15) and nerve bundles formed by the axons of these
neurons (solid and dashed lines) (Palkovits & Brownstein, 1989).
Neurons containing catecholamines including DA have been first iden-

tified on sections with the Falck–Hillarp formaldehyde histofluorescent
technique in the mid-60th (Dahlström & Fuxe, 1964). Basing on this tech-
nique, a number of clusters of DA-containing neurons were mapped in the
brain and designated as A1, A2, . . ., A16 groups. Later, this mapping has
been confirmed and slightly improved (Fig. 4.3) (Palkovits & Brownstein,
1989) by using more advanced techniques such as the Falck–Hillarp
histofluorescent technique combined with catecholamine spectral analysis
and microsurgical lesions of the brain, the glyoxylic acid histofluorescence
method (Björklund & Lindvall, 1984), autoradiography following adminis-

tration of radiolabeled catecholamines (Bosler & Calas, 1982), and immuno-
staining of enzymes of catecholamine synthesis, mostly TH (Hökfelt et al.,
1984; Palkovits & Brownstein, 1989) or DA (Okamura, Kitahama,
Nagatsu, & Geffard, 1988). Morphological data on the location of the
DA-ergic centers in the brain were eventually enriched with physiological
evidence of their involvement in neural and neuroendocrine regulations of a
wide range of physiological functions (Deutch & Roth, 2003). According to
the data accumulated beginning from the initial detection of fluorescence
catecholamine neurons in the 1960s up to the present, the DA-ergic neurons
should be defined as those having specific machinery for DA turnover and
two enzymes of DA synthesis from L-tyrosine (TH and AADC) and the
DAT and VMAT2 (Fig. 4.2). However, VMAT2 is a semispecific marker
of DA-ergic neurons, being also an attribute of other monoaminergic neu-
rons (Weihe et al., 2006).
In addition to DA-ergic neurons, those partly expressing the DA-ergic
phenotype were found in the brain of adult mammals from the late 1980s
onward (Meister et al., 1988; Okamura, H., Kitahama, K., Mons,
et al., 1998; Okamura, H., Kitahama, K., Nagatsu, et al., 1998; Okamura,
H., Kitahama, K., Raynaud, et al., 1998; Skagerberg et al., 1988;
Ugrumov, 2008, 2009). Most of them possessed only one of the enzymes
of DA synthesis, TH or AADC (Ahmed, Northcutt, & Lonstein, 2012;
Ikemoto, Nagatsu, Kitahama, et al., 1998; Ikemoto, Nagatsu, Nishimura,
Nishi, & Arai, 1998; Jaeger, Albert, Joh, & Reis, 1983; Jaeger et al.,
1984; Kontostavlaki et al., 2006; Marsais & Calas, 1999; Marsais,
Parmentier, Terao, Taxi, & Calas, 2002), while lacking the DAT and
VMAT2 (Hoffman et al., 1998; Lorang, Amara, & Simerly, 1994; Weihe
et al., 2006). The same neurons were observed in the developing brain in
ontogenesis (Balan et al., 2000; Ershov, Ugrumov, Calas, Krieger, &
Thibault, 2002; Ershov, Ugrumov, Calas, Makarenko, et al., 2002;
Karasawa, Arai, Isomura, Nagatsu, & Nagatsu, 1997; Nagatsu et al., 1990,
1996). Finding of the neurons partly expressing DA-ergic phenotype raised
the question of their functioning and functional significance. Moreover,
identification of DA-ergic neurons by the presence of only one among three
specific molecular markers—DA (Björklund & Lindvall, 1984; Dahlström &
Fuxe, 1964; Okamura, Kitahama, Nagatsu, et al., 1988), TH (Hökfelt et al.,
1984), and the DAT- or DA-specific uptake (Bosler & Calas, 1982;
Hoffman et al., 1998)—that are still used in literature can no longer be
applicable.
The goal of this chapter is to summarize our own and literature data on
the development, location, functioning, functional significance, and regula-
tion of the neurons partly expressing the DA-ergic phenotype.
2. NEURONS PARTLY EXPRESSING DA-ERGIC

PHENOTYPE IN ONTOGENESIS
2.1. Specific characteristics
Numerous neurons partly expressing the DA-ergic phenotype are widely
distributed in the developing brain in ontogenesis, as shown mostly in
rodents and primates first with three-dimensional reconstruction of the spa-
tial distribution of the neurons mono-immunostained for TH or AADC and
then with double-immunostaining of both enzymes. It is noteworthy that a
part of the neurons immunoreactive for one of the enzymes of DA synthesis
disappears or becomes undetectable as development proceeds, in rats during
the perinatal period. Such transient populations of monoenzymatic
TH-immunoreactive neurons were found in the shrew anterior olfactory
nucleus (Nagatsu et al., 1990), human olfactory bulbs (Verney, Gaspar,
Febvret, & Berger, 1988), mouse raphe nucleus (Karasawa et al., 1997), rat stri-
atum (Sorokin et al. unpublished), human supraoptic and paraventricular nuclei
(Panayotacopoulou, Raadsheer, & Swaab, 1994), etc. (Table 4.1). In most brain
regions, these neurons first increased in number, reaching a peak, and then grad-
ually disappeared (Berger et al., 1985; Nagatsu et al., 1990; Sorokin et al.,
unpublished; Verney et al., 1988). The data in the preceding text are considered
as either a manifestation of the TH transient expression in differentiating neu-
rons or a degeneration of the TH-containing neurons. A reduction of TH syn-
thesis and hence of the intraneuronal content of the TH-immunoreactive
material to an undetectable level is also possible (Karasawa et al., 1997).
Apart of monoenzymatic TH neurons, monoenzymatic AADC neurons
are also widespread in the developing brain that was thoroughly examined in
rats (Jaeger & Teitelman, 1992). These neurons first appear in the rat brain at
the 15th embryonic day, being gathered together to a large cluster in the
intermediate region of the lateral diencephalic vesicle. Later, this accumu-
lation gives rise to a number of smaller groups, distributed in the brain
according to the ventrodorsal gradient. First, the ventral groups located in
the lateral hypothalamic area (D11) and in the anterior hypothalamus,
medial to the medial forebrain bundle (D14), become detectable with
immunocytochemistry. Then, the dorsal groups located in the dorsal mes-
encephalon (D5) and in the midhypothalamus appear. They extend rostrally
towards the bed nucleus joining in the anterior neuron cluster adjacent to
Table 4.1 Transient populations of monoenzymatic TH neurons (cell bodies or fibers)

or transient expression of TH in neurons of the developing brain in mammals
Period of ontogenesis/ Authors (animal, colocalization,
Brain region maximal concentration þ/)
Raphe n. P (1–14) Karasawa et al., 1997 (shrew,
serotonin, þ)
Anterior E16–puberty/P9–P12 Nagatsu et al., 1990 (mouse, þ)
olfactory n.
Olfactory E26 and culture for 14 or Izvolskaia, Duittoz, Ugrumov, &
placodes 18 days Tillet, 2006 (sheep, GnRH, )
Suprachiasmatic P2–P21/P10 Ugrumov, Tixier-Vidal, et al.,
n. 1989 (rat, þ); Beltramo et al., 1994
(rat, þ); Battaglia, Beltramo,
Thibault, Krieger, & Calas, 1995
(rat, þ)
Nasal area— 6–7 GW Verney, el Amraoui, & Zecevic,
olfactory bulbs 1996 (human, GnRH, þ)
Supraoptic n., 37 GW–14 postnatal Panayotacopoulou et al., 1994
paraventricular months/39 GW–3 (human, þ)
n. postnatal months
Bed n. of stria E17–puberty/P5 Verney et al., 1988 (rat,
terminalis somatostatin, substance P, þ)
E17–P7/E17–E19 Sorokin et al. unpublished (rat, )
Central n. of E17–puberty/P5 Verney et al., 1988 (rat,
amygdala somatostatin, substance P, þ)
Medial P7–29/P14–P21 Nagatsu et al., 1996 (mouse, þ)
geniculate n.
Neocortex P8–P24/P12 Berger, Verney, Gaspar, & Febvret,
1985 (rat, )
Striatum E17–P7/E21 Sorokin et al., unpublished (rat, )
þ, direct evidence derived either from no superposition of monolabeled TH and AADC neuronal
populations on adjacent sections or immunostaining of only one of the enzymes of DA synthesis when
using double-immunolabeling technique. , indirect evidence or suggestion. E, embryonic day; GW,
gestational week; n., nucleus; P, postnatal day. Modified from Ugrumov (2009).
the lateral preoptic area (D11) and the neuron accumulation located just
beyond the anterior commissure (D14).
In the rat fetuses from the 16th to the 19th prenatal day, a number of
additional clusters of monoenzymatic AADC neurons become detectable.
They are located in the dorsal mesencephalon (D4) and the dorsal
diencephalon (D6, D7, and D10) including the dorsomedial nucleus (D12),
thalamus (D7), and the lateral habenular nucleus (D6). During the first post-
natal week, additional five clusters of AADC-immunoreactive neurons (D2,
D3, D8, D9, and D13) become visible (Jaeger & Teitelman, 1992).
2.2. Distribution of “monoenzymatic” neurons in the brain

2.2.1 Mediobasal hypothalamus
Information about the location of monoenzymatic neurons in different regions
of the developing brain is sparse and fragmentary except the hypothalamus,
especially the mediobasal hypothalamus (MBH). The latter consists of the arcu-
ate nucleus (AN), a place of location of cell bodies of the DA-producing neu-
rons, and the median eminence (ME), a place of the axon projections towards
the capillaries of the hypophysial portal circulation. TH-immunoreactive neu-
rons were first detected in the rat AN by mono-immunostaining at the 17th–
18th fetal days. They were a priori considered by the authors as bienzymatic
DA-ergic neurons (Daikoku, Kawano, Okamura, Tokuzen, & Nagatsu,
1986; Ugrumov, Taxi, Tixier-Vidal, Thibault, & Mitskevich, 1989), which
was wrong. Indeed, as shown later, AADC-containing neurons first appear
in the developing AN only 2 days later (Fig. 4.4) (Balan et al., 2000; Ershov,
Ugrumov, Calas, Makarenko, et al., 2002), thereby proving that all the initial
TH-immunoreactive neurons were in fact monoenzymatic in nature.
First, three-dimensional computer reconstruction of the space distribu-
tion of mono-immunostained TH neurons and AADC neurons (Balan et al.,
2000) and, then, the use of the double-immunolabeling technique for cou-
nting of monoenzymatic and bienzymatic neurons in the AN of rats at the
20th–21st prenatal day (Ershov, Ugrumov, Calas, Krieger, et al., 2002;
Ershov, Ugrumov, Calas, Makarenko, et al., 2002) have shown that the
entire population of the neurons expressing enzymes of DA synthesis rep-
resented 49% of monoenzymatic TH neurons, the same portion of
monoenzymatic AADC neurons, and only 1% of bienzymatic most proba-
bly DA-ergic neurons (Fig. 4.5). Indeed, there was almost no overlapping in
the distribution of mono-immunostained TH neurons and mono-
immunostained AADC neurons in the AN of rat fetuses: the former neuron
population mostly occupied the ventrolateral area of the AN, while the latter
neuron population and bienzymatic neurons were located in the dorsomedial
area (Fig. 4.4) (Balan et al., 2000). Later, these data were confirmed by using
double-immunostaining for TH and AADC (Ershov, Ugrumov, Calas,
Krieger, et al., 2002; Ershov, Ugrumov, Calas, Makarenko, et al., 2002).
Figure 4.4 Tyrosine hydroxylase (TH)-immunoreactive neurons (A) and aromatic L-

amino acid decarboxylase (AADC)-immunoreactive neurons (B) (mono-
immunolabeling), and the schematic representation of their distribution in the arcuate
nucleus (AN) of rat fetuses on the 21st day of intrauterine development (C). AN, arcuate
nucleus; DM, dorsomedial region of the AN; ME, median eminence; VL, ventrolateral
region of the AN; III, 3rd ventricle. Modified from Balan et al. (2000) and Ugrumov (2008).
According to the double-immunolabeling study, the total number of

monoenzymatic TH neurons in the rat AN decreased progressively from
the end of fetal life to adulthood, resulting in a reduction of this neuron pop-
ulation for about 40% (Fig. 4.5). These timing changes may be explained by
the establishment of the inhibitory control of TH synthesis in ontogenesis
(Ugrumov, 2002) or by apoptosis of a part of monoenzymatic TH neurons
that is less probable (Ershov, Ugrumov, Calas, Makarenko, et al., 2002). In
contrast to monoenzymatic TH neurons, a population of the
monoenzymatic AADC neurons was maintained almost at the same level
from their first appearance at the end of fetal life to adulthood, while the
number of bienzymatic neurons increased abruptly (by 65-fold), mostly over
the perinatal period (Fig. 4.5). From the inverse relation between the
x102
10
A
Number of TH neurons/AN
8
0
E21 P9 Adult
2
x10
14
B
Number of AADC neurons/AN
12
10
0
E21 P9 Adult
2
x10
12
Number of TH and AADC neurons/AN
C
10
0
E21 P9 Adult
Figure 4.5 The number of monoenzymatic neurons expressing TH (A), AADC (B), and of
bienzymatic neurons (C) in the ventrolateral (dotted line) and dorsomedial (solid line)
regions of the AN of male rats at the 21st embryonic day (E21), the 9th postnatal day
(P9), and in adulthood. Mean S.E.M., *P < 0.05. Modified from Ershov, Ugrumov, Calas,
Krieger, et al. (2002).
number of the monoenzymatic TH neurons and bienzymatic neurons in

ontogenesis, it indirectly follows that, with time, at least a part of the initially
monoenzymatic TH-expressing neurons begin to coexpress AADC,
thereby giving rise to bienzymatic neurons. However, even in adulthood,
the total population of monoenzymatic neurons is as numerous as that of
bienzymatic neurons (Fig. 4.5) (Ershov, Ugrumov, Calas, Krieger, et al.,
2002). Although postnatally, the monoenzymatic and bienzymatic neurons
are distributed throughout the AN, the same zonality in their location was
evident in postnatal rats as in fetuses. Namely, most monoenzymatic TH
neurons are accumulated in the ventrolateral AN, whereas the
monoenzymatic AADC neurons and bienzymatic neurons are gathered
together in the dorsomedial AN (Ershov, Ugrumov, Calas, Krieger,
et al., 2002). Despite that TH and AADC are expressed not only in
DA-ergic neurons but also in any other catecholaminergic neurons (norad-
renergic and adrenergic), it was repeatedly reported that only DA-ergic neu-
rons are an attribute of the AN (Girault & Greengard, 2004).
When studying differentiation of the neurons expressing enzymes of DA
synthesis in the developing AN, special attention was usually paid to axonal
projections and interneuronal relationships, which are considered as indirect
morphological indicators of the neuron functioning and functional signifi-
cance. Indeed, most axons of the neurons of the AN are projected to the
external zone of the ME (Fig. 4.4A and B) where they terminate either
in the vicinity or on the primary portal plexus of the hypothalamo-
hypophysial portal circulation. This is an axonal pathway for the delivery
of adenohypophysiotropic neurohormones first to the portal circulation
and then to the pituitary (Ugrumov, 1991).
Nerve fibers, which were initially detected in the developing ME with
histofluorescence of DA or mono-immunostaining of TH, were a priori
considered by many authors as DA-ergic in nature (Ibata et al., 1982;
Ugrumov, Tixier-Vidal, Taxi, Thibault, & Mitskevich, 1989), which appar-
ently was not always the case. Only further development of the double-
immunostaining of the enzymes of DA synthesis combined with confocal
microscopy first made it possible to distinguish monoenzymatic TH fibers,
monoenzymatic AADC fibers, and bienzymatic fibers. It was shown that the
concentration of monoenzymatic TH axons, that is, the number of axons
per unit of the volume of the ME, is maximal in rats at the end of prenatal
life, whereas it decreased gradually during the postnatal period (Fig. 4.6),
simultaneously with the loss of monoenzymatic TH cell bodies in the
AN (Fig. 4.5A) (Ershov, Ugrumov, Calas, Makarenko, et al., 2002).
x104
12 ∗
Density of fibers/unity of ME area 10 ∗

∗
∗
8
6 ∗
0
E21 P9 Adult
Figure 4.6 Density of TH fibers (empty column), AADC fibers (dotted column), and
bienzymatic fibers (streaked column) in the median eminence (ME) of male rats at
the 21st embryonic day (E21), the 9th postnatal day (P9), and in adulthood. Modified
from Ershov, Ugrumov, Calas, Makarenko, et al. (2002).
In contrast to the monoenzymatic TH axons, the concentration of the

monoenzymatic AADC fibers increased progressively in the rat ME from
the end of fetal life to adulthood (Fig. 4.6) (Ershov, Ugrumov, Calas,
Makarenko, et al., 2002), though the number of monoenzymatic AADC neu-
rons (cell bodies) in the AN was maintained at the same level (Fig. 4.5B)
(Ershov, Ugrumov, Calas, Krieger, et al., 2002). This seeming contradiction
can be explained either by ramification of axonal projections of
monoenzymatic AADC neurons of the AN or by the ingrowth to the ME
of monoenzymatic AADC axons originated outside of the AN (Jaeger
et al., 1984). Concentration of bienzymatic fibers also increased progressively
in the ME during ontogenesis (Fig. 4.6) but, in this case, simultaneously with
the increase of the number of bienzymatic cell bodies in the AN (Fig. 4.5C).
These data suggest that an essential part of bienzymatic fibers in the ME belong
to the neurons of the AN (Ershov, Ugrumov, Calas, Makarenko, et al., 2002),
though some of them can be projected from the outside of the AN.
2.2.2 Suprachiasmatic nucleus

Apart of the AN, the suprachiasmatic nucleus (SCN), a pacemaker of circadian
rhythms in mammals (Honma et al., 2012), is rich in monoenzymatic neurons,
cell bodies, and fibers. Indeed, the developing SCN of young rats contains
numerous monoenzymatic AADC neurons coexpressing vasopressin and rare
monoenzymatic TH neurons. The former neuron population is maintained in

adulthood, whereas the latter disappears (Battaglia et al., 1995).
In addition to monoenzymatic AADC cell bodies, the developing SCN
contains a network of noncatecholaminergic TH-immunoreactive fibers
(Beltramo et al., 1994; Ugrumov, Tixier-Vidal, et al., 1989). These fibers
first appear in the rat SCN on the 2nd postnatal day. Thereafter, the number
of TH-immunoreactive fibers increased abruptly until reaching maximum
on the 10th postnatal day (Fig. 4.7), followed by their loss by prepuberty
(Beltramo et al., 1994; Ugrumov, Tixier-Vidal, et al., 1989). These fibers
were shown to lack AADC, the DAT, and serotonin (Battaglia et al.,
1995; Beltramo et al., 1994), being TH monoenzymatic in nature.
Most TH-immunoreactive fibers of the SCN belong to a sparse popu-
lation of small monoenzymatic TH neurons located in the vicinity of the
SCN, mainly in the ventral portion of the anterior hypothalamic nucleus
(Mirochnik, Makarenko, & Ugryumov, 2002). This neuron population first
appears in rats just after birth, a little bit earlier than their processes grow in
the SCN. The neurons (cell bodies) increased in number rapidly for two
Figure 4.7 Innervation of the suprachiasmatic nucleus (SCN) in intact rats at the 2nd, 10th,
and 21st postnatal days by TH-immunoreactive fibers (A, B, left side) (Ugrumov, Tixier-Vidal,
et al., 1989), which are resistant to 6-hydroxydopamine (C, left side) that is in contrast to
TH-immunoreactive fibers of the paraventricular nucleus (C, right side) (Beltramo et al.,
1994; Ugrumov, Tixier-Vidal, et al., 1989). 6-OHDA; 6-hydroxydopamine; OC, optic chiasma;
P, postnatal day; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; TH, tyrosine
hydroxylase; III, 3rd ventricle. Shaded columns, TH-immunoreactive fibers in intact rats;
solid columns, TH-immunoreactive fibers in rats following the intraventricular injection
of 6-OHDA. *P < 0.05, the number of TH-immunoreactive fibers in the SCN at P10 and
P21 compared to that at P2 and P10, respectively. ♦P < 0.05, the number of
TH-immunoreactive fibers in the PVN in intact rats compared to that in rats following
the intraventricular injection of 6-OHDA.
postnatal weeks and then almost disappeared by prepuberty, simultaneously

with the loss of the monoenzymatic TH fibers in the SCN. In addition to
monoenzymatic TH neurons of the anterior hypothalamic nucleus, rare
monoenzymatic TH neurons located in the periphery of the SCN or in
the adjacent periventricular nucleus can also contribute to a transient inner-
vation of the SCN (Battaglia et al., 1995).
2.2.3 Magnocellular nuclei

In addition to the neurons of the AN and SCN, the magnocellular
vasopressinergic neurons possess TH (Abramova, Marsais, Calas, Thibault, &
Ugrumov, 2002). Although the magnocellular neurons of the supraoptic
and paraventricular nuclei are generated and initiate vasopressin synthesis rather
early in ontogenesis, in rats around the 15th fetal day (Ugrumov, 2002), rare
neurons begin to coexpress TH significantly later, in rats during the neonatal
period (Abramova, Calas, & Ugrumov, 2011). In contrast to rodents, in
humans, TH is coexpressed in numerous vasopressinergic and oxytocinergic
neurons over the entire studied period of ontogenesis, at least from the 37th
gestational week to the 14th postnatal month with the maximum extended
from the 39th gestational week to the 3rd postnatal month. The TH expression
by the maximal number of vasopressinergic and oxytocinergic neurons just
before and after parturition is considered as an adaptive reaction, for example,
to hypoxia (Panayotacopoulou et al., 1994).
2.2.4 Striatum
Striatum and adjacent brain regions, for example, the periventricular
area next to the lateral ventricles and the bed nucleus of stria terminalis,
appear to be particularly rich in transient populations of monoenzymatic
TH neurons in ontogenesis (Fig. 4.8). Indeed, accumulations of
TH-immunoreactive but AADC-immunonegative neurons were detected
in the bed nucleus of stria terminalis of rats from the 17th fetal day to puberty
with a maximum on the 5th postnatal day (Verney et al., 1988).
Rare TH-immunoreactive most probably monoenzymatic neurons
were observed in the rat striatum with mono-immunolabeling first on the
17th fetal day, followed by an increase of this population until reaching max-
imum 4 days later. Then, these neurons almost disappeared over the perina-
tal period (Sorokin et al., unpublished). Although the fate of transient
populations of TH-immunoreactive neurons in the striatum and in other
brain regions during ontogenesis is of particular interest, so far, no attempts
were undertaken to solve this problem. Nevertheless, it might be suggested
A B C E21
E21
NE
ST
AC
20µm
OT
D
E19
Figure 4.8 TH-immunoreactive neurons in the striatum (A, B) and neural epithelium around
lateral ventricles (B, C) in rats at the 21st embryonic day and in the bed nucleus of stria
terminalis at the 19th embryonic day (D). AC, anterior commissure; E, embryonic day; OT,
optic tracts; ST, striatum; NE, neural epithelium; BNST, bed nucleus of stria terminalis.
that the loss of TH-immunoreactive neurons in the striatum and other brain
regions in ontogenesis is a result of either the establishment of the inhibitory
control of either TH expression or the neuron migration to other brain
regions, which is less probable.
Thus, numerous neurons partly expressing the DA-ergic phenotype,
mostly monoenzymatic TH neurons lacking the DAT, are widely distributed
throughout the developing brain and disappear to a large extent by puberty.
3. NEURONS PARTLY EXPRESSING DA-ERGIC

PHENOTYPE IN ADULTHOOD
3.1. Specific characteristics
Already from the initial studies of the neurons mono-immunostained for
the enzymes of DA synthesis, it followed that, in addition to the brain
regions with a complete spatial overlapping of the populations of TH-
immunoreactive neurons and AADC-immunoreactive neurons, for exam-
ple, in the substantia nigra, there were brain areas with partial or no
overlapping of these populations (Okamura, Kitahama, Nagatsu, et al.,

1988; Okamura, Kitahama, Raynaud, et al., 1988; Zoli, Agnati, Tinner,
Steinbusch, & Fuxe, 1993). These data first suggested that the brain contains
monoenzymatic neurons in addition to bienzymatic DA-ergic neurons.
Interestingly, the monoenzymatic neurons predominate in some brain areas
not only in ontogenesis (see earlier) but also in adulthood. Indeed, the num-
ber of TH-immunoreactive neurons in the hypothalamus manifold exceeds
that of DA-containing (histofluorescent) neurons (Björklund & Lindvall,
1984; Van den Pol, Herbst, & Powell, 1984). The development of the
double-immunolabeling technique made it first possible to prove an exis-
tence of monoenzymatic neurons expressing either TH or AADC, which
are widely distributed in the brain in all so far studied adult mammals,
rodents, primates, etc. (Tables 4.2 and 4.3).
Although there is no doubt of the existence of non-DA-ergic
monoenzymatic neurons (Ahmed et al., 2012; Jaeger et al., 1983;
Okamura et al., 1985; Ugrumov, 2009), some data also suggest the existence
of non-DA-ergic neurons expressing both enzymes of DA synthesis but lac-
king the DAT and VMAT2 (Hoffman et al., 1998; Weihe et al., 2006). For
instance, occasional magnocellular vasopressinergic neurons of the supraop-
tic nucleus in humans coexpress TH and AADC though lacking the DAT
(Kontostavlaki et al., 2006). Furthermore, numerous bienzymatic neurons
in the AN of adult rats apparently do not coexpress the DAT, as only a half
of the whole population of bienzymatic neurons degenerated after the
administration of 6-hydroxydopamine, a toxin that is captured in the
DA-ergic neurons by the DAT (Ershov, Ugrumov, Calas, Krieger, &
Thibault, 2005). The inability of most bienzymatic neurons as of any other
neurons in the AN to capture DA and hence 6-hydroxydopamine was con-
firmed by the observation of almost no radioactive labeling of the AN fol-
lowing the intraventricular administration of tritiated DA (Bosler &
Calas, 1982).
Thus, the neurons partly expressing DA-ergic phenotype represented
mostly by monoenzymatic neurons lacking the DAT and VMAT2, though
the existence of bienzymatic neurons lacking the DAT also cannot be
excluded.
3.2. Distribution of monoenzymatic neurons in the brain

3.2.1 Mediobasal hypothalamus
Despite a wide distribution of monoenzymatic neurons in the brain, those
located in the hypothalamus and striatum have attracted in literature
Table 4.2 Monoenzymatic TH neurons, cell bodies, and fibers, in the brain of intact
adult mammals
Brain region Authors (animal, colocalization, þ/)
Cell bodies and fibers
Arcuate n. Okamura, Murakami, Chihara, Nagatsu, & Ibata, 1985
(rat, GH-RH, ); Meister et al., 1988 (rat, þ); Okamura,
Kitahama, Nagatsu, et al., 1988 (rat, ); Tinner et al.,
1989 (rat, choline acetyltransferase, ); Komori, Fujii, &
Nagatsu, 1991 (human, þ); Zoli et al., 1993 (rat, );
Ershov, Ugrumov, Calas, Krieger, et al., 2002 (rat, þ);
Karasawa et al., 2007 (monkey); Ahmed et al., 2012
(prairie vole, þ)
Supraoptic n. Panayotacopoulou et al., 1994 (human, vasopressin,
oxytocin, ); Kitahama et al., 1998 (human, vasopressin, þ);
Abramova et al., 2002 (rat, vasopressin, ); Marsais et al.,
2002 (rat, vasopressin, )
Paraventricular n. Panayotacopoulou et al., 1994 (human, vasopressin,
oxytocin, ); Kitahama et al., 1998 (human, þ); Marsais
et al., 2002 (rat, vasopressin, ); Karasawa et al., 2007
(monkey, vasopressin, þ);
Kontostavlaki et al., 2006 (human, vasopressin, þ)
Accessory n. Kitahama et al., 1998 (human, vasopressin, þ); Marsais
et al., 2002 (rat, vasopressin, )
Anterior olfactory n. Nagatsu et al., 1990 (mouse, ); Kitahama et al., 1998
(human, þ)
Olfactory tubercles Nagatsu et al., 1990 (human, þ); Karasawa et al., 2007
(monkey, þ)
Olfactory bulbs Weihe et al., 2006 (rat, mouse, monkey, þ)
Preoptic SCN Karasawa et al., 2007 (þ)
Periventricular n. Battaglia et al., 1995 (rat, þ); Karasawa et al., 2007
(monkey, þ); . Ahmed et al., 2012 (prairie vole, þ)
Medial preoptic area Ahmed et al., 2012 (prairie vole, þ)
Lateral preoptic area Vincent & Hope, 1990 (hamster, þ)
Zona incerta Skagerberg et al., 1988 (rat, ); Ahmed et al., 2012
(prairie vole, þ)
Striatum Tashiro, Sugimoto, et al., 1989 (rat, ); Lopez-Real,
Rodriguez-Pallares, Guerra, & Labandeira-Garcia, 2003
Continued
adult mammals—cont'd
(rat, þ); Cossette, Lévesque, et al., 2005 (rat, DAT,
Nurr1, GAD-65, calretinin, ); Tandé et al., 2006
(monkey, DAT, Nurr1, GAD-65, calretinin, )
Caudate n. Dubach et al., 1987 (monkey, ); Betarbet et al., 1997
(monkey, ); Cossette, Lecomte, et al., 2005 (human,
DAT, ); Huot, Lévesque, & Parent, 2007 (human, )
Putamen Dubach et al., 1987 (monkey, ); Betarbet et al., 1997
(monkey, ); Cossette, Lecomte, et al., 2005 (human,
DAT, ); Huot et al., 2007 (human, DAT, )
N. accumbens Ikemoto, Nagatsu, Kitahama, et al., 1998 (human, )
Amygdala Ahmed et al., 2012 (prairie vole, þ)
Bed n. of the stria Ahmed et al., 2012 (prairie vole, þ)
terminalis
Cortex (frontal, Weihe et al., 2006 (rat, monkey, þ)
occipital)
Anterior cingulate Ikemoto et al., 1999 (human, þ)
cortex
Substantia nigra, Ikemoto, Nagatsu, Nishimura, et al., 1998 (human, );
compact zone Karasawa et al., 2007 (monkey,þ); Ahmed et al., 2012
(prairie vole, þ)
Ventral tegmental area Ahmed et al., 2012 (prairie vole, þ)
Ventral and dorsal Ikemoto, Nagatsu, Nishimura, et al., 1998 (human,)
tegmental area
Globus pallidus Komori et al., 1991 (human, þ)
Periaqueductal gray Karasawa et al., 2007 (monkey, þ)
matter
Medial longitudinal Karasawa et al., 2007 (monkey, þ)
fasciculus
N. tractus solitarius Weihe et al., 2006 (monkey,þ); Karasawa et al., 2007
(monkey, þ)
N. of horizontal limb Vincent & Hope, 1990 (hamster, þ)
of diagonal band
Pericentral n. of inferior Vincent & Hope, 1990 (hamster, þ)
colliculus
Lateral parabrachial n. Vincent & Hope, 1990 (hamster, þ); Weihe et al., 2006
(monkey, þ)
Dorsal motor n. of Vincent & Hope, 1990 (hamster, þ); Weihe et al., 2006
vagus (rat, mouse, monkey, þ)
Substantia innominata Vincent & Hope, 1990 (hamster, þ)
Area postrema Weihe et al., 2006 (monkey, þ)
Cerebellum Sakai, Fujii, Karasawa, Arai, & Nagatsu, 1995 (mouse, )
Habenula Weihe et al., 2006 (rat, þ)
Nerve fibers
Median eminence Ershov, Ugrumov, Calas, Makarenko, et al., 2002 (rat, þ)
Posterior lobe Abramova, Calas, Thibault, & Ugrumov, 2000 (rat,
vasopressin, þ)
þ, direct evidence derived either from no superposition of monolabeled TH and AADC neuronal
using double-immunolabeling technique. , indirect evidence or suggestion. Modified from Ugrumov
(2009).
particular attention that is explained by the crucial role of these brain

regions in neuroendocrine regulations and the control of motor function,
respectively. In adulthood, like in ontogenesis, the hypothalamic AN,
SCN, and magnocellular nuclei were shown to be particularly rich in
monoenzymatic neurons that became evident even in the initial studies
when using mono-immunostaining of TH and AADC. In the AN of adult
rats, most TH-mono-immunostained neurons were located in the ventro-
lateral region of the AN, while the AADC-mono-immunostained neurons
predominated in the dorsomedial region (Fig. 4.5) (Okamura, Kitahama,
Nagatsu, et al., 1988; Okamura, Kitahama, Raynaud, et al., 1988), showing
low overlapping in between and hence suggesting that an essential number
of the neurons are monoenzymatic (Okamura, Kitahama, Nagatsu, et al.,
1988; Okamura, Kitahama, Raynaud, et al., 1988; Zoli et al., 1993). This
suggestion was further proved by using double-immunolabeling of TH and
AADC (Ershov, Ugrumov, Calas, Krieger, et al., 2002; Ershov, Ugrumov,
Table 4.3 Monoenzymatic AADC neurons, cell bodies, and fibers, in the brain of intact
adult mammals
Brain region Authors (animal, þ/)
Cell bodies and fibers
Arcuate n. Okamura, Kitahama, Mons, et al., 1988 (rat, ); Ershov,
Ugrumov, Calas, Krieger, et al., 2002 (rat, þ); Karasawa
et al., 2007 (monkey, þ); Ahmed et al., 2012 (prairie
vole, þ)
Suprachiasmatic n. Jaeger et al., 1983 (rat, þ); Karasawa et al., 1994 (shrew þ);
Battaglia et al., 1995 (rat, þ); Kitahama et al., 1998 (human, þ);
Novak & Nunez, 1998 (hamster, þ); Ishida et al., 2002 (rat, þ)
Dorsomedial n. Jaeger et al., 1984 (rat, ); Kitahama et al., 1998 (human, );
Karasawa et al., 2007 (monkey, þ)
Premammillary n. Jaeger et al., 1984 (rat, ); Karasawa et al., 1994 (shrew, þ);
Kitahama et al., 1998 (human, )
Mammillary n. Karasawa et al., 2007 (monkey, þ)
Zona incerta Skagerberg et al., 1988 (rat, ); Ahmed et al., 2012 (prairie
vole, þ)
Medial preoptic area Vincent & Hope, 1990 (hamster, þ); Kitahama et al., 1998
(human, ); Ahmed et al., 2012 (prairie vole, þ)
Zona incerta Kitahama et al., 1998 (human, )
Lateral hypothalamic Kitahama et al., 1998 (human, ); Karasawa et al., 2007
area (monkey, þ)
Anterior Karasawa et al., 2007 (monkey, þ)
hypothalamic n.
Bed n. of the stria Kitahama et al., 1998 (human, ); Ahmed et al., 2012
terminalis (prairie vole, þ)
Stria medullaris Karasawa et al., 2007 (monkey, þ)
Striatum Tashiro, Kaneko, et al., 1989 (rat, )
Medullary reticular n. Lopez-Real et al., 2003 (rat, þ); Karasawa et al., 2007
(monkey, þ)
Caudate n. Ikemoto et al., 1997 (human, )
Putamen Ikemoto et al., 1997 (human, )
Amygdala Ahmed et al., 2012 (prairie vole, þ)
Table 4.3 Monoenzymatic AADC neurons, cell bodies, and fibers, in the brain of intact
Brain region Authors (animal, þ/)
N. accumbens Ikemoto et al., 1997 (human, )
Anterior cingulate Ikemoto et al., 1999 (human, þ)
cortex
Multiassociative Gaspar et al., 1987 (rat, )
cortex
Unimodal associative Gaspar et al., 1987 (rat, )
cortex
Substantia nigra, Ikemoto, Nagatsu, Nishimura, et al., 1998 (human, );
compact zone Karasawa et al., 2007 (monkey, þ); Ahmed et al., 2012
(prairie vole, þ)
Ventral tegmental area Ikemoto, Nagatsu, Nishimura, et al., 1998 (human, þ);
Ahmed et al., 2012 (prairie vole, þ)
Pretectal n. Ikemoto, Nagatsu, Nishimura, et al., 1998 (human, þ)
Lateral parabrachial n. Vincent & Hope, 1990 (hamster, þ)
Cerebellum Sakai et al., 1995 (mouse, )
Nerve fibers
Median eminence Ershov, Ugrumov, Calas, Makarenko, et al., 2002 (rat, þ)
þ, direct evidence derived either from no superposition of mono-labeled TH and AADC neuronal
using double-immunolabeling technique. , indirect evidence or suggestion. Modified from Ugrumov
(2009).
Calas, Makarenko, et al., 2002). Moreover, according to the double-

immunolabeling data, most bienzymatic neurons and monoenzymatic
AADC neurons are located in the dorsomedial region of the AN
(Fig. 4.6). Similar distribution of bienzymatic neurons and monoenzymatic
TH and AADC neurons in the AN is typical for other so far studied mam-
mals (Tables 4.1 and 4.2) (Ahmed et al., 2012; Karasawa et al., 2007;
Kitahama et al., 1998; Komori et al., 1991). From quantitative immunocy-
tochemical image analysis, it follows that the whole population of the neu-
rons expressing enzymes of DA synthesis in the AN of adult rats consists of
approximately 16% of monoenzymatic TH neurons, 46% of monoenzymatic
AADC neurons, and 38% of bienzymatic neurons (Fig. 4.5) (Ershov,
Ugrumov, Calas, Krieger, et al., 2002). These data strongly support the idea
in the preceding text that the population of monoenzymatic neurons at least

in some brain regions prevails that of bienzymatic neurons, thereby empha-
sizing functional importance of the former.
Although a certain progress was made at studying the monoenzymatic
neurons and their topographic relations by using double-immunostaining
of the enzymes of DA synthesis in combination with confocal microscopy
(Ershov, Ugrumov, Calas, Makarenko, et al., 2002; Ugrumov, 2009), res-
olution of this technique was insufficient to determine whether the
bienzymatic and monoenzymatic neurons are different in their subcellular
organization and what are the intimate topographic relations in between.
Therefore, electron microscopy combined with a double-immunolabeling
of TH and AADC was recently used to approach these issues (Ugrumov
et al., unpublished). This technique made it first possible to recognize
with certainty bienzymatic most probably DA-ergic neurons and
monoenzymatic neurons expressing TH or AADC (cell bodies) in the
MBH, as other neurons expressing these enzymes, noradrenergic, adrener-
gic, and serotoninergic neurons, are located outside the hypothalamus. As to
nerve fibers in the MBH, only axons and dendrites immunoreactive for TH
and immunonegative for AADC were considered as true monoenzymatic
fibers. The axons double-immunostained or mono-immunostained for
AADC can belong not only to DA-ergic neurons of the AN but also to other
catecholaminergic neurons (DA-ergic, noradrenergic, and adrenergic) and
serotoninergic neurons, respectively, which are located outside of the
AN, for example, in the diencephalon and brain stem (Hökfelt, 2010;
Palkovits & Brownstein, 1989).
According to our electron microscopic data, all types of the neurons
expressing enzymes of DA synthesis in the rat AN appeared to be similar
in shape, size, and subcellular composition (Ugrumov et al., unpublished).
Bienzymatic and monoenzymatic neuron cell bodies contain moderately
developed Golgi complex, well-developed granular endoplasmic reticulum,
numerous ribosomes (polysomes), and mitochondria (Fig. 4.9A), altogether
suggesting their high metabolic activity (Ugrumov et al., unpublished). In
addition to organelles, the neurons possess inclusions, mostly small dense-
cored vesicles, primary lysosomes, or precursors of secretory granules. Many
neurons also contain large vacuoles sometimes with membrane conglomer-
ates inside (Ugrumov et al., unpublished), which probably represent
autophagosomes serving for degradation of cellular organelles (Boellaard,
Schlote, & Hofer, 2004; Singh et al., 2009).
PVS
CL
C
∗
Figure 4.9 Double-immunolabeling at the electron microscopic level of TH (electron-

dense gold–silver particles, small arrow) and AADC (peroxidase, large arrow): (A)
double-immunostained neuron (cell body) in the AN (star); (B) AADC-immunoreactive
axonal terminal (large arrow) in the ME abutting on the capillary of the primary portal
plexus; (C) apposition between the TH-immunoreactive nerve fiber (snowflake) and
AADC-immunoreactive axonal terminal (large arrow). CL, capillary lumen; PVS,
perivascular space.
Special attention was paid to topographic relation of bienzymatic and

monoenzymatic neurons, cell bodies, and processes, in between in the
AN, and with the hypophysial capillary portal plexus (axovascular contacts)
in the ME. According to the double-immunolabeling study in confocal
microscope, all types of the neurons expressing enzymes of DA synthesis
were often seen in close topographic relations and even in appositions,
suggesting functional interactions in between. Besides simple appositions,
Figure 4.10 Axosomatic specialized-like contact (arrowhead) between monoenzymatic

TH-immunoreactive axon (white) and monoenzymatic AADC-immunoreactive cell body
(gray) in the AN of the adult rat (Ershov et al., 2002b). Confocal microscopy: double-
labeling of TH and AADC (A) and mono-labeling of AADC (B) and TH (C). AADC, aromatic
L-amino acid decarboxylase; TH, tyrosine hydroxylase. Dotted line, an outlined AADC-
immunoreactive neuron.
specialized-like junctions were observed with confocal microscopy (Ershov,

Ugrumov, Calas, Makarenko, et al., 2002). They represented by ramified
monoenzymatic TH axons spreading along cell bodies of monoenzymatic
AADC neurons (Fig. 4.10) (Ershov, Ugrumov, Calas, Makarenko, et al.,
2002). Appositions between the monoenzymatic TH neurons and AADC
neurons were observed not only at the level of cell bodies in the AN but
also, most often, at the level of axonal terminals in the ME (Ershov,
Ugrumov, Calas, Makarenko, et al., 2002). Moreover, numerous
monoenzymatic TH axons and AADC axons were shown to terminate in
vicinity or on the basal lamina of the primary capillary plexus of the hypo-
physial portal circulation, giving rise to the so-called axovascular contacts.
The observations of appositions between the neurons expressing enzymes
of DA synthesis including monoenzymatic neurons, and of axovascular
contacts obtained in confocal microscope, were further proved with elec-

tron microscopy (Fig. 4.9B and C). These data strongly support the idea
about functional interactions between monoenzymatic and bienzymatic
neurons, on the one hand, and show an axonal pathway for the delivery
of neurohormones and their releasable metabolites towards the hypophysial
portal circulation, on the other.
In addition to appositions between the neurons expressing enzymes of
DA synthesis, bienzymatic neurons were often seen to be synaptically inner-
vated by immunonegative nonmonoaminergic axons. Neurotransmission
within these synapses is most probably provided by neuropeptides.
3.2.2 Anterior hypothalamus

Suprachiasmatic nucleus. The SCN, a pacemaker of the circadian rhythmic
activity, as the AN contains numerous monoenzymatic neurons. Most of
them coexpressing AADC and vasopressin are located in the ventromedial
and ventrolateral regions of the nucleus (D13) (Fig. 4.11; Table 4.2)
(Battaglia et al., 1995; Ishida et al., 2002; Jaeger et al., 1983; Kitahama
Figure 4.11 Schematic representation of the distribution of TH-immunoreactive neu-

rons (A) and AADC-immunoreactive neurons (B) in the hamster suprachiasmatic nuclei
(Novak & Nunez, 1998). The neurons are represented as dots. OC, optic chiasma; SCN,
suprachiasmatic nucleus; III, 3rd ventricle.
et al., 1998). Besides, occasional monoenzymatic TH neurons were detected

in the periphery of the SCN (Fig. 4.10) (Battaglia et al., 1995; Novak &
Nunez, 1998). Functional significance of the expression of individual
enzymes of DA synthesis in this nucleus remains unclear.
Magnocellular nuclei. Over two last decades, it has been repeatedly
reported that occasional vasopressinergic neurons of the hypothalamic
supraoptic nucleus, the paraventricular nucleus, and “accessory” nuclei of
intact animals are capable of coexpressing TH (Table 4.2). In addition to
vasopressinergic neurons, oxytocinergic neurons coexpressing TH were
detected in the hypothalamic magnocellular nuclei but, so far, only in
humans (Panayotacopoulou et al., 1994). In rodents, the number
vasopressinergic neurons coexpressing TH multiplies under functional stim-
ulation, for example, at salt loading or hypergravity (Abramova et al., 2002;
Marsais et al., 2002; Ugrumov, 2002; Ugrumov, Pronina, et al., 2002). In
vasopressinergic neurons of rodents, TH is diffusively distributed through-
out cell bodies, dendrites, and axons projecting to the pituitary posterior
lobe (Abramova et al., 2000).
Besides the hypothalamic regions described in the preceding text, the
monoenzymatic TH neurons and AADC neurons are widely distributed
in other hypothalamic areas and all through the brain in its extension from
the olfactory bulbs rostrally to the brain stem caudally (Tables 4.2 and 4.3).
3.2.3 Striatum
Although many immunocytochemical studies have been devoted to the
nigrostriatal DA-ergic system of intact mammals, only few of them managed
to detect rare neurons expressing enzymes of DA synthesis in the striatum
and in the adjacent limbic system in rodents, for example, in the nucleus
accumbens (Tables 4.2 and 4.3) (Betarbet et al., 1997; Tashiro, Kaneko,
et al., 1989; Tashiro, Sugimoto, et al., 1989). In fact, the number of
TH-immunoreactive neurons in rats does not exceed 20 per striatum
(Tashiro, Sugimoto, et al., 1989), whereas AADC-immunoreactive neurons
are even fewer (Lopez-Real et al., 2003; Tashiro, Kaneko, et al., 1989).
Rare TH-immunoreactive neurons (1–3 per 40 mm thick section) were also
found with mono-immunolabeling in the nucleus accumbens (Table 4.2).
Most striatal neurons expressing enzymes of DA synthesis are
monoenzymatic, though some of them appear to coexpress both enzymes
(Lopez-Real et al., 2003). Monoenzymatic TH-immunoreactive neurons
and AADC-immunoreactive neurons, 10–20 mm in diameter, round or oval
Figure 4.12 Schematic representation of the distribution of TH-immunoreactive neu-

rons (A) and AADC-immunoreactive neurons (B) in the rat striatum after
6-hydroxydopamine-induced degeneration of nigral DA-ergic neurons (Lopez-Real
et al., 2003). ac, anterior commissure; cc, corpus callosum; v, lateral ventricle.
in shape, give rise to the axons and one or two spiny dendrites (Tashiro,
Kaneko, et al., 1989; Tashiro, Sugimoto, et al., 1989). No zonality in the
distribution of the striatal monoenzymatic neurons was observed in rodents
in major studies, though, according to some authors, most monoenzymatic
TH neurons and AADC neurons are located dorsally, in the subcallosal area
of the striatum (Fig. 4.12) (Lopez-Real et al., 2003).
There are many more striatal neurons expressing enzymes of DA synthe-
sis in primates (monkeys and humans) compared to rodents (Tables 4.2 and
4.3) (Betarbet et al., 1997; Cossette, Lecomte, & Parent, 2005; Lopez-Real
et al., 2003). Indeed, the number of striatal TH-immunoreactive neurons,
though varying from monkey to monkey, is in average 40–75 per 40 mm
thick section (Betarbet et al., 1997; Dubach et al., 1987; Tandé et al., 2006).
Almost all TH-immunoreactive neurons (99%) were shown to coexpress
the DAT and, therefore, were considered as DA-ergic neurons, though
nobody attempted yet to detect in these neurons AADC using a triple-
labeling technique. In monkeys, the mono-immunostained TH neurons
are located mainly at the periphery of the caudate nucleus and putamen
(Betarbet et al., 1997; Dubach et al., 1987; Tandé et al., 2006). Dorsally,
these neurons are distributed along the boundary of the striatum under
the corpus callosum. A ventral population of striatal TH-immunoreactive
neurons extends towards the population of DA-ergic neurons in the ventral
forebrain. TH-immunoreactive neurons are also distributed along the lateral
edge of the putamen, in the neuropil, and in the adjacent white matter.
The striatal TH-immunoreactive neurons in monkeys are usually round

or oval in shape and small in size (10–18 mm) having from two to five aspiny
processes. Moreover, a few (<1%) larger (15–25 mm) multipolar neurons
with spiny processes were observed (Betarbet et al., 1997).
In addition to monkeys, mono-immunolabeled TH neurons and AADC
neurons were also observed in the striatum of humans in norm, that is, with-
out clinical or pathological signs of neurological or psychiatric disorders
(Tables 4.2 and 4.3). TH-immunoreactive neurons are distributed through-
out the striatum, being more numerous in the putamen than in the caudate
nucleus (Cossette, Lecomte, et al., 2005; Cossette, Parent, & Lévesque,
2004; Huot et al., 2007). Few TH neurons were also detected in the ventral
nucleus accumbens (Ikemoto, Nagatsu, Kitahama, et al., 1998). The number
of TH-immunoreactive neurons in the human striatum varies from several
hundred to several thousand (Cossette, Lecomte, et al., 2005).
More than a half of TH-IR neurons (58%) consist of the medium-sized
(20–24 mm) or small-sized (10–15 mm) cell bodies, axons, and varicose aspiny
dendrites (Cossette, Lecomte, et al., 2005; Huot et al., 2007). Although
monoenzymatic TH neurons with aspiny dendrites are distributed throughout
the caudate nucleus and putamen, they predominate in the ventral striatum.
The minor population of TH-immunoreactive neurons (6.5%) is represented
by large neurons (25–35 mm) with spiny dendrites. In contrast to aspiny
TH-immunoreactive neurons, the spiny TH neurons were detected in the
caudate nucleus but not in the putamen. In addition to typical neurons,
few TH-IR cells are similar in appearance to glial cells having round or ovoid
cell body (10–12 mm) with up to ten aspiny processes. They attach to the
striatal bridges, which link to the caudate nucleus and the putamen rostrally.
AADC neurons in humans exceed those in rodents in number and size
(Ikemoto et al., 1997). Monoenzymatic AADC-IR neurons are distributed
in the entire rostrocaudal extension of the striatum and in the nucleus
accumbens (Table 4.3). Most numerous AADC neurons are located in
the nucleus accumbens, less numerous neurons is a characteristic of puta-
men, and the least population of AADC-immunoreactive neurons is
attached to the caudate nucleus. Most AADC neurons are fusiform, bipolar,
or multipolar in shape; medium to large in size (15–30 mm); and having thick
ramified dendrites.
Thus, the neurons partly expressing the DA-ergic phenotype, mostly
monoenzymatic neurons lacking the DAT, are widely distributed all
through the brain in adulthood, being in some brain areas even more
numerous than the DA-ergic neurons.
4. FUNCTIONING OF MONOENZYMATIC NEURONS

Finding of non-DA-ergic neurons partly expressing enzymes of DA
synthesis and their wide distribution in the brain raised a question about their
functioning and functional significance.
4.1. Monoenzymatic neurons expressing TH

It has been shown that TH is represented in monoenzymatic neurons by
the same isoforms as in catecholaminergic neurons (Marsais et al., 2002), being
in most neurons enzymatically active and capable of producing L-DOPA as a
final synthetic product. This conclusion follows from the observations in
the ventrolateral AN containing mostly monoenzymatic TH neurons of
(i) the neuron population mono-immunostained for TH and L-DOPA
but immunonegative for AADC and DA (Meister et al., 1988; Okamura,
Kitahama, Mons, et al., 1988; Okamura, Kitahama, Nagatsu, et al., 1988),
(ii) TH-expressing neurons with no DA histofluorescence even after systemic
administration of L-DOPA (Zoli et al., 1993), and (iii) unexpectedly high level
of L-DOPA in the rat AN enriched with TH-expressing neurons and almost
lacking bienzymatic neurons at the end of fetal life (Melnikova et al., 1999;
Ugrumov, Melnikova, Ershov, Balan, & Calas, 2002). In addition to the
AN, the neurons containing L-DOPA as a final synthetic product were found
in other brain regions including the substantia nigra, ventral tegmental region,
and the raphe nucleus (Mons, Tison, & Geffard, 1989).
Along with monoenzymatic neurons possessing enzymatically active TH,
in some neurons, this enzyme is not capable of conversion of L-tyrosine in
L-DOPA probably because of the lack of tetrahydrobiopterin, the
enzyme cofactor, synthesized by guanosine triphosphate cyclohydrolase
I (GTPC). Indeed, a number of monoenzymatic TH neurons such as hypo-
thalamic magnocellular vasopressinergic neurons (Marsais et al., 2002), cere-
bellar Purkinje cells (Sakai et al., 1995), the neurons of the developing anterior
olfactory nucleus (Nagatsu et al., 1990), and the neurons of the geniculate
nucleus (Nagatsu et al., 1996) were shown to be immunonegative for
L-DOPA and GTPC. Therefore, the Weihe’s et al. (2006) proposal to call
the monoenzymatic TH neurons as “dopaergic” may be applied only to
the neurons with enzymatically active TH. However, it cannot be excluded
that GTPC and L-DOPA are produced in all monoenzymatic TH neurons
but, in some of them, in such small amounts, which cannot be detected with
traditional methods. Indeed, a coexpression of TH and GTPC genes in some
vasopressinergic neurons of the human supraoptic nucleus was recently shown

by using a laser microdissection combined with real-time PCR (Kontostavlaki
et al., 2006). Still, it remains unclear whether the GTPC expression in mag-
nocellular vasopressinergic neurons is the species-specific phenomenon or, in
fact, a resolution of the in situ hybridization, and immunocytochemistry is
insufficient to detect GTPC mRNA or GTPC protein in the vasopressinergic
neurons of rodents.
For understanding of the basic principles of functioning of
monoenzymatic TH neurons, it is important to recognize whether they
coexpress the DAT. At least a part of monoenzymatic TH neurons, for
example, of the AN, apparently do not coexpress the DAT. This was proven
by showing in the neurons of the ventrolateral area of the rat AN containing
mainly monoenzymatic TH neurons of (i) no DAT mRNA and the DAT
protein (Fig. 4.13) (Hoffman et al., 1998), (ii) a low potential for DA uptake
(Bosler & Calas, 1982; Melnikova et al., 1999; Moore et al., 1985), and
(iii) no degeneration of monoenzymatic TH neurons after the administra-
tion of 6-hydroxydopamine (Ershov et al., 2005). Low level of the DAT
expression and the neuron inability for the DA uptake in the AN by contrast
with some other DA-producing brain regions, for example, substantia nigra,
is usually explained by a different local role of DA. Indeed, DA released into
the synaptic cleft in neuropil, for example, in the striatum, should be rapidly
inactivated after its action as a neurotransmitter. Conversely, DA released
from the axons in the ME should be delivered to the portal blood vessels
for further distant action on the target pituitary cells, lactotrophs, as a neu-
rohormone. However, this interpretation, though being generally accepted,
should be taken with caution in light of the fact that the pituitaries of trans-
genic mice lacking the DAT gene fail to develop lactotrophs (Bossé
et al., 1997).
Besides the AN, monoenzymatic TH neurons lacking the DAT, cell
bodies, and fibers are widely distributed in other brain regions. They were
found in the SCN, periventricular nucleus (A14), preoptic area (A15),
supraoptic nucleus, posterior hypothalamic nucleus (A11), bed nucleus of
the stria terminalis (A15), rostral liner nucleus, rostral periaqueductal gray
(A11), and interpeduncular nucleus (Beltramo et al., 1994; Lorang et al.,
1994; Ugrumov, Tixier-Vidal, et al., 1989). Nevertheless, it remains to
be uncertain whether the DAT is an attribute of the striatal monoenzymatic
TH neurons. Although almost all the striatal TH-immunoreactive neurons
were shown to possess the DAT, no attempts were undertaken so far to rec-
ognize whether these neurons coexpress AADC being DA-ergic in nature,
III
ME
III
ME
III
ME
Figure 4.13 TH, dopamine transporter (DAT) and vesicular monoamine transporter
type 2 (VMAT2) expressions in the AN: (A) TH expression in the dorsomedial (arrow)
and ventrolateral (arrowhead) regions of the AN; (B) DAT expression in the dorsomedial
but not in the ventrolateral region of the AN; (C) VMAT2 expression in the dorsomedial
region but not in the ventrolateral region (C) (Hoffman et al., 1998). ME, median emi-
nence; III, third ventricle. Bar scale ¼ 150 mm.
as it was suggested by the authors (Cossette, Lecomte, et al., 2005; Cossette,

Lévesque, & Parent, 2005; Tandé et al., 2006).
Although L-DOPA is generally considered as a final synthetic product in
most monoenzymatic TH neurons, the mechanisms of its intraneuronal
storage and release remain unclear. Anyway, from the observation of the
L-DOPA release under the membrane depolarization from the fetal AN
(Melnikova et al., 1999) containing numerous monoenzymatic TH neurons

but almost lacking bienzymatic neurons (Ershov, Ugrumov, Calas, Krieger,
et al., 2002), it indirectly follows that L-DOPA is stored in secretory granules
(dense-cored vesicles) and released by exocytosis. This idea appears to con-
tradict to the statement that monoenzymatic TH neurons lack VMAT2, and
therefore, L-DOPA cannot be stored in secretory granules and released by
exocytosis (Hoffman et al., 1998; Weihe et al., 2006). However, it cannot
be excluded that the cytosolic secretory products like DA and L-DOPA can
be released by a nonexocytotic mechanism (Hoffman et al., 1998; Weihe
et al., 2006). This is in line with a finding of nonexocytotic DA release from
the midbrain DA-ergic neurons via the DAT in VMAT2-deficient trans-
genic mice (Fon et al., 1997). The nonexocytotic mechanism of the L-
DOPA release, if it exists, should differ from that of DA as most
monoenzymatic TH neurons lack the DAT (Hoffman et al., 1998).
L-DOPA synthesis in the monoenzymatic TH neurons as a final synthetic
and releasable product raised a question about its functional significance.
Three suggestions were proposed in literature to solve this issue. According
to the first one, L-DOPA plays a role of the intercellular signal, neurotrans-
mitter, or neurohormone as evidenced by (i) L-DOPA release from the stri-
atum via Ca2þ-dependent mechanism; (ii) L-DOPA stimulatory influence
via b-adrenoreceptors on noradrenaline release; (iii) dose-dependent
L-DOPA influence via D2 receptors on DA release and AADC activity
in the nigral DA-ergic neurons (Fisher, Biggs, Eradiri, & Starr, 2000;
Misu, Kitahama, & Goshima, 2003); (iv) close topographic relations of
monoenzymatic TH neurons with the presumptive targets for L-DOPA,
for example, vasoactive intestinal polypeptide-producing neurons in the
SCN (Battaglia et al., 1995); and (v) a termination of monoenzymatic
TH-immunoreactive axons (Ershov, Ugrumov, Calas, Makarenko, et al.,
2002) or L-DOPA-immunoreactive fibers (Misu et al., 2003) on the primary
capillary plexus of the hypophysial portal circulation. Although the hypoth-
esis about the L-DOPA role as an intercellular signal is rather attractive, it
should be taken with caution, as the principal supporting data were obtained
in the experiments with a “complete” pharmacological inhibition of AADC
(Misu et al., 2003). In fact, it is rather difficult or even impossible to inhibit
completely the enzymes of synthesis of classical neurotransmitters.
According to the second and more speculative hypothesis, L-DOPA
plays a role of the intracellular signal-controlling synthesis and release of
coexpressing neurotransmitters. This hypothesis is supported indirectly by
the fact that TH is coexpressed in the neurons producing different types
of neurotransmitters including g-aminobutyric acid, neurotensin, somato-

statin, growth hormone-releasing hormone, dinorphin, galanin, vasopres-
sin, substance P, serotonin, choline acetyltransferase, and the key enzyme
of acetylcholine synthesis (Everitt et al., 1986; Karasawa et al., 1997;
Marsais et al., 2002; Okamura et al., 1985; Tinner et al., 1989; Verney
et al., 1988). According to the third hypothesis, L-DOPA contributes to
the regulation of the neuron differentiation during the so-called critical
period of ontogenesis that might explain the meaning of the transient
expression of TH and possible time-limited L-DOPA production in onto-
genesis, in rats during the perinatal period (Verney et al., 1996; Izvolskaia
et al., 2006). However, all the attempts undertaken so far for detection of
L-DOPA in transient populations of TH-expressing neurons were unsuc-
cessful (Karasawa et al., 1997).
Thus, the most widespread TH monoenzymatic neurons produce
L-DOPA as a final synthetic product, which is stored and discharged under
adequate physiological stimuli.
4.2. Monoenzymatic neurons expressing AADC

In contrast to TH, AADC is an unspecific enzyme that is capable of conver-
sion of L-DOPA in DA not only in monoenzymatic AADC neurons but also
in catecholaminergic neurons and serotoninergic neurons (Ishida et al.,
2002; Karasawa et al., 1994). In all cases, L-DOPA should be captured in
monoenzymatic AADC neurons by the semispecific membrane transporter
of large neutral amino acids, an attribute of different cells including neurons
(Fernández, Torrents, Zorzano, Palacı́n, & Chillarón, 2005). DA synthesis
in monoenzymatic AADC neurons was proved by showing the DA accu-
mulation in these neurons after the L-DOPA systemic administration that
was observed in the SCN (D13) (Ishida et al., 2002), premammillary nucleus
(D18), pretectal nucleus (D15), and the nucleus of the solitary tract (D2)
(Karasawa et al., 1994).
Although DA synthesis in monoenzymatic AADC neurons has been
proven, mechanisms of its storage and release remain unknown. Taking into
account that these neurons do not express VMAT2, DA is believed to be
accumulated and released by a nonexocytotic mechanism (Weihe et al.,
2006). Indeed, a large amount of DA was discharged under membrane depo-
larization from the AN of rat fetuses (Melnikova et al., 1998, 1999) containing
numerous monoenzymatic AADC neurons but almost lacking bienzymatic
DA-ergic neurons (Ershov, Ugrumov, Calas, Krieger, et al., 2002). The
monoenzymatic AADC neurons appear to lack the DAT that indirectly fol-
lows from a failure of 6-OHDA, neurotoxin of DA-ergic neurons, to provoke
their degeneration (Ershov et al., 2005). Nevertheless, an existence of
monoenzymatic AADC neurons expressing the DAT cannot be excluded.
Indeed, it was not defined so far whether the neurons having the DAT but
lacking VMAT2 and TH (Hoffman et al., 1998) are capable of coexpressing
AADC. The hypothetic neurons of this kind coexpressing AADC and DAT
could serve for capturing and storage of extracellular DA similar to non-
serotoninergic neurons coexpressing the serotonin membrane transporter
and AADC (Ugrumov, Taxi, Steinbusch, Tramu, & Mitskevich, 1989).
Despite that monoenzymatic AADC neurons were found a long time
ago (Jaeger et al., 1984), still little is known about their functional signifi-
cance. Therefore, any information about colocalization of AADC with neu-
rotransmitters or neuromodulators is of importance. So far, only
colocalization of AADC with vasopressin in neurons of the SCN has been
definitely shown (Jaeger et al., 1983). A number of other monoenzymatic
AADC neurons were also suggested to coexpress neuropeptides that indi-
rectly followed from the spatial overlapping of the populations of
monoenzymatic AADC neurons, on the one hand, and of enkephalin-
producing neurons in the lateral parabrachial nucleus (D3), substance
P-producing, and neurotensin-producing neurons in the nucleus of the sol-
itary tract (D2), on the other (Jaeger et al., 1984). From the observation that
the monoenzymatic AADC neurons are in close topographic relations with
cerebral blood vessels (Ugrumov, Taxi, Steinbusch, et al., 1989), it indirectly
follows that these neurons may synthesize DA from L-DOPA circulated in
the blood (Karasawa et al., 1994; Melnikova et al., 2006).
In addition to DA synthesis from L-DOPA, the monoenzymatic AADC
neurons should be capable of decarboxylation of amino acids, phenylalanine,
tyrosine, and tryptophan directly to the corresponding trace amines, phenyleth-
ylamine, tyramine, and tryptamine (Jaeger et al., 1983, 1984; Kitahama
et al., 1998).
Thus, widespread monoenzymatic AADC neurons synthesize DA as a final
product, which is stored and discharged under adequate physiological stimuli.
4.3. Monoenzymatic neurons expressing either TH or aromatic

L-amino acid as a functional unit
4.3.1 Cooperative synthesis of DA in ontogenesis
Most authors who studied monoenzymatic TH neurons and AADC neurons
have attempted to recognize functional significance of each population
separately, but only according to our methodology; the neurons expressing

individual complementary enzymes of DA synthesis were considered as a
functional unit. According to our hypothesis, L-DOPA produced in
monoenzymatic TH neurons is released to the intercellular space and then
captured with the membrane transporter of large neutral amino acids in
monoenzymatic AADC neurons for its further conversion to DA
(Ugrumov, 2008, 2009; Ugrumov, Melnikova, et al., 2002). DA apparently
can be synthesized by monoenzymatic TH neurons and AADC neurons even
if they are topographically separated, as L-DOPA was shown to diffuse via
intercellular clefts for a long distance (Schneider, Rothblat, &
DiStefano, 1994).
The rat AN at the end of prenatal life containing more than 99% of
monoenzymatic TH neurons and AADC neurons and <1% of bienzymatic
neurons (Balan et al., 2000; Ershov, Ugrumov, Calas, Krieger, et al., 2002)
appeared to be an excellent model to test our hypothesis of cooperative syn-
thesis of DA by monoenzymatic neurons. According to the early ex vivo and
cell culture studies, the DA concentration in the fetal AN highly exceeded
(Melnikova et al., 1999) that in other fetal brain regions and in the entire
brain containing a large number of DA-ergic (bienzymatic) neurons
(Coyle & Henry, 1973). Furthermore, it was found that the amount of
DA produced in the AN of rat fetuses was high enough to provide the inhib-
itory control of the adenohypophysial prolactin secretion prenatally
(Melnikova et al., 1998) as in adulthood (Ben-Jonathan & Hnasko, 2001;
Moore et al., 1985). This conclusion followed from the observation of
the increased concentration of prolactin in plasma and reduced concentra-
tion of prolactin in the pituitary in rat fetuses after systemic administration to
pregnant mothers of haloperidol, an inhibitor of DA receptors (Melnikova
et al., 1998). Taking together the data in the preceding text, one could con-
clude that DA is synthesized in the AN of fetal rats by the monoenzymatic
TH- and AADC-expressing neurons in cooperation, as the production of
such a large amount of DA by occasional bienzymatic neurons seems to
be improbable.
Although the in vivo, ex vivo, and in vitro data in the preceding text are in
favor of cooperative synthesis of DA in the fetal AN, they still are considered
as indirect evidence since the input of the minor population of bienzymatic
neurons to DA synthesis has not been defined and, hence, cannot be
completely ignored. Our attempts to obtain a pure population of
monoenzymatic neurons in the fetal AN by removing the bienzymatic neu-
rons with 6-hydroxydopamine were unsuccessful because of the low level of
the DA and, hence, 6-hydroxydopamine uptake in this peculiar brain region

(Ugrumov et al., unpublished). Therefore, the only way to obtain direct evi-
dence of cooperative synthesis of DA by monoenzymatic neurons was to
prevent or at least to slow down the L-DOPA transport from the
monoenzymatic TH neurons to monoenzymatic AADC neurons with no
influence on DA synthesis in bienzymatic DA-ergic neurons. If the hypoth-
esis is correct, this action should provoke a decrease of DA synthesis in
monoenzymatic neurons but not in bienzymatic neurons, thereby resulting
in a drop of DA content in the AN as a whole. Three approaches could
be theoretically used to solve this problem: (i) the inhibition of L-DOPA
release from the monoenzymatic TH neurons, (ii) the inactivation of L-
DOPA in the extracellular space, and (iii) the inhibition of the L-DOPA
uptake by the monoenzymatic AADC neurons. The last approach has been
finally used (Ugrumov, Melnikova, Lavrentyeva, Kudrin, & Rayevsky,
2004). The presumptive L-DOPA uptake by monoenzymatic AADC neu-
rons was prevented by the administration of exogenous L-tyrosine as a com-
petitive inhibitor of the membrane transporter under static incubation of the
cell suspension of the AN of fetal rats (Fig. 4.14). Cell suspension of the sub-
stantia nigra of the same fetuses containing mostly DA-ergic neurons was
used as a control. Overall amount of DA in the incubation medium and cell
extracts after the incubations was taken as an index of the rate of DA syn-
thesis. As expected, L-tyrosine has provided opposite effects on DA synthesis
in the substantia nigra and AN of rat fetuses, stimulating DA synthesis in the
former as a substrate of the enzymatic reaction and reducing DA synthesis in
the latter as a competitive inhibitor of the L-DOPA uptake by
monoenzymatic AADC neurons (Fig. 4.14). These data were considered
as a direct proof of DA synthesis by monoenzymatic TH neurons and
AADC neurons in tandem (Ugrumov et al., 2004).
4.3.2 Cooperative synthesis of DA in adulthood

Although cooperative synthesis of DA by the monoenzymatic neurons in
fetuses has been proven, this should not be a priori the case for adulthood,
as the composition of monoaminergic neurons, cell bodies, and fibers was
changed significantly in ontogenesis. Indeed, the total population of
monoenzymatic neurons in the AN of adult rats was almost halved while
that of bienzymatic (DA-ergic) neurons manifold increased compared to
rat fetuses (Fig. 4.5) (Ershov, Ugrumov, Calas, Makarenko, et al., 2002).
Moreover, over the perinatal period of ontogenesis, the MBH received
numerous axonal projections from DA-ergic, noradrenergic, adrenergic,
Tyr (–)
0.6
Dopamine (ng/sample)
A B Tyr (+)
Dopaminergic neurons 0.5
0.4
TH & 0.3
AADC 0.2 *
0.1
L-Tyrosine Dopamine
0
Monoenzymatic neurons
1.0
Dopamine (ng/sample)
C *
TH AADC 0.8
0.6
L-DOPA
0.4
L-Tyrosine 0.2
0
Figure 4.14 Schematic representation (A) and results (B, C) of the experiment showing
that the competitive inhibition of the L-DOPA transporter with L-tyrosine (A) resulted in
the decrease of DA synthesis in cell suspension of the AN of rats at the 21st embryonic
day (B) containing mostly monoenzymatic TH neurons and AADC neurons and in the
increase of DA synthesis in cell suspension of the substantia nigra of the same fetuses
(C) containing mostly DA-ergic neurons (Ugrumov et al., 2004). Tyr (), incubation of cell
suspension in the absence of L-tyrosine; Tyr (þ), incubation of cell suspension in the
presence of L-tyrosine. AADC, aromatic L-amino acid decarboxylase; TH, tyrosine hydrox-
ylase. Filled oval, neutral amino acid and L-DOPA transporter. Mean S.E.M., *P < 0.05.
and serotoninergic neurons located in other brain regions, mostly in the

mesencephalon and brain stem. Therefore, it has been suggested that, in
adulthood, L-DOPA produced in monoenzymatic TH neurons can be
converted to DA not only in monoenzymatic AADC neurons as in fetuses
but also in any other AADC-expressing neurons (cell bodies and fibers),
DA-ergic, noradrenergic, adrenergic, and serotoninergic (Ugrumov, 2009).
The same methodology was used to prove DA cooperative synthesis in
adulthood as in fetuses (Ugrumov et al., 2004). Namely, L-DOPA uptake
from the extracellular space to AADC-expressing neurons was prevented
with a large neutral L-amino acid as a competitive inhibitor of the L-DOPA
membrane transporter (Misu et al., 2003; Uchino et al., 2002; Vieira-
Coelho & Soares-Da-Silva, 1998). However, a design of the experiment
was substantially modified. First, slices of the MBH of adult rats
(Ugrumov et al., unpublished) were used instead of the cell suspension of
the MBH of rat fetuses for providing better preservation of the neurons
and maintaining to a certain extent the native interneuronal relationships.
Then, L-tyrosine, used earlier as a competitive inhibitor of the L-DOPA
Leu (–)
A 0.8
Dopamine (ng/tissue)
B Leu (+)
DA synthesis
0.6
TH & AADC 0.4
*
0.2
DA
L-Leucine L-DOPA 0
0.3 C
Dopamine (ng/ml)
TH AADC
0.2
L-DOPA
0.1
*
L-Leucine
0
Figure 4.15 Schematic representation (A) and results (B, C) of the experiment showing
that the competitive inhibition of the L-DOPA transporter with L-leucine (A) resulted in
the decrease of DA synthesis in slices (B) of the mediobasal hypothalamus of adult rat
and incubation medium following incubation perfusion (C) (Ugrumov et al.,
unpublished ). Leu (), incubation of slices in the absence of L-leucine; Leu (þ), incuba-
tion of slices in the presence of L-leucine. AADC, aromatic L-amino acid decarboxylase;
TH, tyrosine hydroxylase. Filled oval, neutral amino acid and L-DOPA transporter. Clear
arrows show a presumptive decrease of DA synthesis in both DA-ergic neurons and
monoenzymatic neurons because of the inhibition of the L-DOPA uptake. Mean
S.E.M., *P < 0.05.
membrane transporter in monoenzymatic AADC neurons of the MBH of

rat fetuses almost lacking bienzymatic (DA-ergic) neurons, was replaced
by L-leucine as the MBH of adult rats contains numerous DA-ergic neurons
along with monoenzymatic neurons (Fig. 4.15). Indeed, L-leucine in con-
trast to L-tyrosine is indifferent to the DA metabolism in DA-ergic neurons.
On the other hand, the absence of L-tyrosine in the incubation medium
should not affect DA production in the MBH, as an intraneuronal reserve
of this amino acid is high enough to maintain DA synthesis at a normal level
for a quite a long time (Büyükuysal and Moğol, 2000).
According to the design of our recent experiment, the slices of the MBH
and substantia nigra obtained from the same adult rats were incubated sep-
arately under static or perfusion incubation with or without L-leucine in the
medium. According to our data, the L-leucine administration did not affect
the DA concentration in slices of the substantia nigra and in the incubation
media under static incubation and perfusion incubation (Ugrumov et al.,
unpublished), as this brain area contains mostly bienzymatic DA-ergic neu-
rons (Kitahama et al., 1998). On the contrary, the L-leucine administration
resulted in a drop of the DA concentration in the incubation medium after

static incubation of the MBH and in a decrease of the DA concentration
both in the slices and the incubation medium after the perfusion incubation
of the MBH (Ugrumov et al., unpublished) possessing numerous mono-
enzymatic neurons (Ershov, Ugrumov, Calas, Krieger, et al., 2002). These
data are considered as direct evidence of DA synthesis in the MBH in adulthood
by monoenzymatic TH neurons in cooperation not only with monoenzymatic
AADC neurons but also with any other neurons expressing AADC, catechol-
aminergic, and serotoninergic neurons (Fig. 4.16) (Arai, Karasawa, Geffard, &
Nagatsu, 1995; Ugrumov, Melnikova, et al., 2002). Indeed, extracellular
L-DOPA captured in the catecholaminergic neurons should promote catechol-
amine synthesis, as the AADC enzymatic activity highly exceeds that of TH.
In turn, the uptake of extracellular L-DOPA to serotoninergic neurons should
initiate DA synthesis (Fig. 4.16). In addition to monoenzymatic AADC
neurons, glial cells and endothelial cells of blood vessels possess enzymatically
active AADC (Hardebo, Emson, Falck, Owman, & Rosengren, 1980;
Juorio, Li, Walz, & Paterson, 1993) being capable of synthesizing DA from
L-DOPA derived either from the monoenzymatic TH neurons or coming
from general circulation (Melnikova et al., 2006).
L-Tyrosine
TH
L-DOPA
TH& TryH&
AADC AADC
AADC
Dopamine
Figure 4.16 Schematic representation of cooperative synthesis of DA by

(i) monoenzymatic neurons expressing complementary enzymes of DA synthetic path-
way (center), (ii) strengthening of DA synthesis in DA-ergic neurons due to L-DOPA pro-
duced in monoenzymatic TH-expressing neurons (left side), (iii) initiation of DA
synthesis in serotoninergic neurons due to L-DOPA produced in monoenzymatic
TH-expressing neurons (right side). AADC, aromatic L-amino acid decarboxylase; TH,
tyrosine hydroxylase; TryH, tryptophan hydroxylase.
Neuron functioning and functional significance in general are believed to

be determined to some extent by interneuronal relationships. Appositions or
even specialized-like junctions between the monoenzymatic TH neurons and
any other neurons expressing AADC observed in our studies first with con-
focal microscopy and then with electron microscopy (Figs. 4.9 and 4.10) are
considered as a manifestation of their functional interaction, serving for safety
transport of L-DOPA from the former to the latter. In addition to close inter-
neuronal relationships, monoenzymatic TH axons, monoenzymatic AADC
axons, and bienzymatic axons were often seen abutting on the primary cap-
illary plexus of the hypophysial portal circulation in the ME (Fig. 4.9).
Although DA, a prolactin-inhibiting neurohormone, has been detected in
portal blood a long time ago (Ben-Jonathan & Hnasko, 2001), still, it is
believed to be produced only in DA-ergic neurons of the MBH. In light
of the evidence of DA cooperative synthesis, DA circulating in portal blood
should be also produced in monoenzymatic TH neurons in tandem with any
other neurons containing AADC. In addition to DA, L-DOPA is assumed to
be released from the monoenzymatic TH axons and delivered to the hypo-
physial portal circulation or general circulation. Circulating L-DOPA was
shown to be involved in DA synthesis in peripheral monoenzymatic cells
expressing AADC, for example, the tubule cells of the kidney (Hayashi,
Yamaji, Kitajima, & Saruta, 1990; Meister, Fried, Holgert, Aperia, &
Hökfelt, 1992), thereby suggesting that cooperative synthesis of DA is not
restricted to the brain neurons but is widespread in the whole organism.
5. CONCLUSION
5.1. Functional significance of monoenzymatic neurons
in norm and pathology
Despite obtaining convincing evidence that monoenzymatic neurons syn-
thesize DA, functional role of this cooperative synthesis in addition to
DA synthesis in DA-ergic neurons remained unclear until recently. We
hypothesized that cooperative synthesis of DA is a compensatory mechanism
activated by functional insufficiency of DA-ergic neurons, for example,
under their chronic stimulation or a partial loss, for example, in neurodegen-
erative diseases like hyperprolactinemia and Parkinson’s disease (Ugrumov,
2008; Ugrumov, Melnikova, et al., 2002). This idea has been further
supported by the fact that, in response to the neurotoxin-induced degener-
ation of 50% DA-ergic neurons and a loss of 50% DA in the AN of adult rats,
0.9% NaCl
60 6-OHDA
A Arcuate B
Prolactin in plasma (ng/ml)

nucleus 50 **
*
40
TH&
TH & AADC AADC
6-OHDA 30
TH&
AADC 20
TH III ventricle TH
10
Median
eminence 0
14th day 45th day
0.5 60
C ** D
Number of neurons/section
Dopamine (ng/mg tissue)
* *
0.4
40
0.3
*
0.2 *
20
0.1
0 0
14th day 45th day TH& TH AADC
AADC
Figure 4.17 Schematic representation of the experiment (A) and results (B–D) showing
that the 6-hydroxydopamine-induced degeneration of 50% bienzymatic (TH and AADC)
neurons in the AN of adult rats (D) is followed first by decreased DA synthesis and
increased prolactin secretion (B, C; 14th day) and then (B, C; 45th day) by the normal-
ization of DA synthesis and prolactin secretion (Ziyazetdinova et al., 2008). Moreover,
the number of monoenzymatic neurons increased significantly (D) (Ershov et al.,
2005). AN, arcuate nucleus; AADC, aromatic L-amino acid decarboxylase; TH, tyrosine
hydroxylase, 6-OHDA, 6-hydroxydopamine. *P < 0.05, for a difference between the
experiment and the control; **P < 0.05, for a difference between selected parameters.
there was an increase of the number of monoenzymatic TH and AADC neu-

rons and the return to normal DA synthesis (Fig. 4.17) (Ershov et al., 2005;
Ziyazetdinova et al., 2008). A similar reaction is a characteristic of the striatal
monoenzymatic neurons, which either appear (Fig. 4.18A–F) (Sorokin et al.,
unpublished) or increase in number after degeneration (Fig. 4.18G) of the
nigral DA-ergic neurons and a depletion of striatal DA (Betarbet et al.,
1997; Cossette, Lecomte, et al., 2005; Meredith et al., 1999; Ugrumov,
2008). In this context, DA synthesis in monoenzymatic neurons is considered
14
12
10
Mean cell no.
6
S6
4 S5
S4
2
S3
Level
0 S2
Week 4+
Week 3 S1
Week 2
Survival Week 1
Figure 4.18 Mono-immunolabeled (A, B, E, F) and double-immunolabeled (C, D) neu-

rons for either TH (A, B, C, E, F) or AADC (D), which appeared in the rat striatum after
6-hydroxydopamine-induced degeneration of nigral DA-ergic neurons (Sorokin et al.,
unpublished). The number of TH-immunoreactive neurons at six rostrocaudal levels
of the rat striatum (S1–S6) 1, 2, 3, and 4 weeks after 6-hydroxydopamine administration
(G) (Meredith et al., 1999).
among most important mechanisms of the brain plasticity, which are activated
in dopamine-deficient neurodegenerative diseases, hyperprolactinemia, and
Parkinson’s diseases, providing their asymptomatic development for a long
time (Ugrumov, 2008).
In addition to monoenzymatic AADC-expressing neurons, any other

AADC-expressing cells including endothelial cells, glial cells, catecholamin-
ergic neurons, and serotoninergic neurons are believed to be capable of cap-
turing extracellular L-DOPA originated from the monoenzymatic
TH-expressing neurons or general circulation (Hardebo et al., 1980;
Juorio et al., 1993). This is probably accompanied by a compensatory
increase in DA synthesis as a final synthetic product in DA-ergic neurons
and as an intermediate synthetic product in noradrenergic and adrenergic
neurons. Moreover, L-DOPA uptake by serotoninergic neurons should ini-
tiate DA synthesis (Fig. 4.16) (Ugrumov, 2008, 2009). The phenomenon in
the preceding text underlies conventional therapy of Parkinson’s disease by
using L-DOPA (Ikemoto, 2002; Mura, Jackson, Manley, Young, & Groves,
1995). Additional indirect evidence of catecholamine synthesis from L-
DOPA in cells expressing AADC but lacking TH was obtained at studying
mice with an inactivated gene for TH. Indeed, prenatal lethality of these
mice was prevented by the systemic administration of L-DOPA (Zhou,
Quaife, & Palmiter, 1995).
Thus, a substantial number of the brain neurons express partly the
DA-ergic phenotype, mostly individual complementary enzymes of DA
synthesis serving to produce DA in cooperation that is apparently a compen-
satory reaction under a failure of DA-ergic neurons.
5.2. Regulation of monoenzymatic neurons

5.2.1 Neural regulation of monoenzymatic neurons
Although monoenzymatic neurons were found a long time ago, there is still
little information about their regulation. Nevertheless, some indirect evi-
dence allowed us to assume that TH expression is under an inhibitory influ-
ence of the afferent innervation, especially of the catecholaminergic fibers
(Ugrumov, 2008, 2009). These observations include (i) the increased num-
ber of TH-immunoreactive vasopressinergic neurons in the rat supraoptic
nucleus following lesion of the medial forebrain bundle (Kiss & Mezey,
1986); (ii) the increased number of TH-immunoreactive vasopressinergic
neurons of the supraoptic nucleus in Brattleboro rats, which by contrast with
normal rats (Kiss & Mezey, 1986) are incapable of synthesizing vasopressin
and receive poor noradrenergic innervation (Schöler & Sladek, 1981);
(iii) the appearance of TH-immunoreactive neurons in the ventrolateral
region of the rat AN following its microsurgical deafferentation (Daikoku
et al., 1986); (iv) a loss of TH-immunoreactive neurons in the striatum
and adjacent brain regions as their synaptic innervation proceeds in
ontogenesis (Ugrumov, 2009) and reappearance of TH-immunoreactive

neurons after the 6-hydroxydopamine-induced DA-ergic denervation of
the striatum (Darmopil, Muñetόn-Gόmez, de Ceballos, Bernson, &
Moratalla, 2008; Sorokin et al., unpublished); and (v) the increased number
of monoenzymatic TH neurons in the AN, striatum, and olfactory bulbs fol-
lowing 6-hydroxydopamine-induced DA-ergic denervation (Ershov et al.,
2005; Tashiro, Kaneko, Nagatsu, Kikuchi, & Mizuno, 1990; Tashiro,
Kaneko, et al., 1989; Tashiro, Sugimoto, et al., 1989).
Direct proof of the hypothesis in the preceding text was obtained so far
only for vasopressinergic neurons of the supraoptic nucleus by showing that
the TH expression is inhibited by noradrenergic afferents and the noradren-
aline influence is mediated via a1-adrenoreceptors (Fig. 4.19) (Abramova
et al., 2011). Indeed, the vasopressinergic neurons of salt-loaded neonatal
rats treated with prazosin, the antagonist of a1-adrenoreceptors, showed
an increase of (i) the number of TH-IR neurons, (ii) the intraneuronal
TH concentration, and (iii) the TH mRNA concentration in
vasopressinergic neurons. Opposite results were obtained using phenyleph-
rine, the agonist of a1-adrenoreceptors (Abramova et al., 2011).
5.2.2 Paracrine and endocrine regulations of monoenzymatic neurons

Neurotrophic or growth factors produced mainly in glial cells appear to be
among the most potent chemical signals stimulating the expression of
enzymes of DA synthesis in non-DA-ergic neurons. This suggestion indi-
rectly follows from the observation of a simultaneous increase in secretion
of neurotrophic factors and the expression of individual enzymes of DA syn-
thesis in the striatal neurons as the degeneration of nigral DA-ergic neurons
proceeds (Nakajima et al., 2001). Indeed, the intensity of both processes
increased continuously until reaching maximum 2–3 weeks after the neu-
rotoxin injection (Meredith et al., 1999; Nakajima et al., 2001). Neuro-
trophic factors are especially potent in the primate brain that is manifested
in a multiple increase of the number of the neurons expressing TH and
the DAT in the striatum of intact old monkeys or in young monkeys follow-
ing DA-ergic denervation of the striatum (Palfi et al., 2002). The fibroblast
growth factor and the brain-derived neurotrophic factor were shown to be
also efficient in stimulation of TH expression in non-DA-ergic neurons in
the denervated striatum, and DA mediates their action. Although the glial-
derived neurotrophic factor and the ciliary neurotrophic factor provide sim-
ilar action on the striatal neurons, they are less potent (Du & Iacovitti, 1995).
Brainstem
Anterior
hypothalamus
Posterior
lobe of
hypophysis
Figure 4.19 Schematic representation of the inhibitory influence of noradrenergic

afferents of the brain stem neurons and noradrenaline (black dots) via a1-receptors
(filled triangle) on TH expression (snowflake) in vasopressinergic neurons of the supra-
optic nucleus. Filled circles, vasopressin secretory granules; filled triangle,
adrenoreceptor. Modified from Ugrumov (2008).
With time, evidence of the direct action of growth factors on the striatal
neurons via specific receptors was accumulated. Indeed, the administration
of neurotrophic factors to the striatal tissue culture resulted in the appearance
of TH-immunoreactive cells, first after 12 h of incubation. Thereafter, the
number of TH-immunoreactive neurons increased rapidly, reaching max-
imum 18 h after the treatment, followed by its gradual decrease for subse-
quent 4 days (Du & Iacovitti, 1995). The glia-derived neurotrophic
factor was shown to provide its action via a multireceptor complex com-
posed of a novel glycosylphosphatidylinositol-anchored glia-derived
neurotrophic factor receptor-a and the receptor tyrosine kinase product
of the c-ret proto-oncogene (Durbec et al., 1996; Trupp, Belluardo,

Funaksohi, & Ibanez, 1997). The expression of this receptor in the developing
brain in rats is maximal during the early postnatal period, simultaneously with
a high level of the TH expression (Sorokin et al., unpublished), followed by a
decrease of both parameters by adulthood (Trupp et al., 1997).
In addition to growth factors, vasopressin seems to be an extracellular
signal providing the inhibitory control of the TH expression in
vasopressinergic neurons. This suggestion is supported by the observation
of the decreased level of TH in the neurons of the magnocellular nuclei
in vasopressin-deficient Brattleboro rats treated with exogenous vasopressin
(Kiss & Mezey, 1986).
A list of chemical signals controlling the expression of the enzymes of
DA synthesis in non-DA-ergic neurons apparently is not limited to vaso-
pressin and neurotrophic factors. Some signals provide different effects when
acting on the neurons in various brain regions or in the same region over
different periods of ontogenesis. For instance, serotonin deficiency pro-
voked by p-chlorophenylalanine, an inhibitor of tryptophan hydroxylase,
resulted in the increase in the number of TH-immunoreactive neurons in
the dorsal motor vagal nucleus (Kitahama, Bérod, Denoyer, & Jouvet,
1987) and in the decrease in TH content in differentiating neurons of the
AN, as shown in vivo and in cell culture (Melnikova, Ugrumov,
Proshlyakova, Calas, & Thibault, 2001). Furthermore, serotonin provides
a long-lasting effect on differentiating TH neurons in the rat AN as a mor-
phogenetic diffusive factor that was manifested by a declining level of TH in
the neurons of the AN in adult rats treated with p-chlorophenylalanine dur-
ing the intrauterine development (Melnikova et al., 2001).
So far, only a few works have provided information about the regulation
of AADC expression in monoenzymatic neurons. Thus, it was shown that
the AADC gene expression and AADC activity in monoenzymatic neurons
of the rat SCN strongly depend on the circadian rhythms being maximal
during daylight hours and minimal over nocturnal period. Although the reg-
ulation of AADC expression in these neurons by extracellular signals is quite
probable, its mechanisms remain unclear (Ishida et al., 2002).
Regulation of monoenzymatic neurons is probably not limited to nerve
afferents and paracrine signals, but is also provided by hormones of the endo-
crine glands. Indeed, it has been repeatedly shown that DA-producing
neurons and TH-expressing neurons of the AN coexpress receptors mostly
for hormones of reproduction, prolactin, progesterone, and estrogens
(Arbogast & Voogt, 1991, 1997; Dufourny, Caraty, Clarke, Robinson,
& Skinner, 2005; Hou, Yang, & Voogt, 2003; Lerant & Freeman, 1998;
McCann et al., 1984; Mitchell et al., 2003; Moore et al., 1985;
Ugrumov, 2008; Warembourg, Varoqueaux, & Poulain, 1993). However,
only Lemoine, Leroy, and Warembourg (2005) have attempted, so far, to
recognize whether these neurons are bienzymatic or monoenzymatic at
studying the hypothalamus of guinea pig with triple labeling of TH, AADC,
and the progesterone receptor. They have demonstrated that the progester-
one receptors are expressed in all monoenzymatic AADC neurons in the
preoptic area and in numerous monoenzymatic and bienzymatic neurons
of the AN. Namely, among the progesterone-expressing neurons in the
AN, 29% of the neurons coexpress only TH, 9% only AADC, and 29% both
TH and AADC.
Thus, the monoenzymatic and bienzymatic neurons and especially the
expression of the enzymes of DA synthesis in these neurons are under the
control of neural afferents, paracrine factors, and hormones of the endocrine
glands.
CONFLICT OF INTEREST
The authors have no conflicts of interest to declare.
ACKNOWLEDGMENTS
The present study was supported by the following grants: programs of the Presidium of the
Russian Academy of Sciences “Basic Sciences for Medicine,” program of the Department of
Physiology and Fundamental Medicine of the Russian Academy of Sciences “Mechanisms of
Integration of Molecular Systems in the Implementation of Physiological Functions,”
RFBR-11-04-01840, RFBR-oriented basic research 11-04-112121, RFBR-
CNRS-10-04-93108, and RFH 12-06-00894.
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CHAPTER FOUR

Brain Neurons Partly Expressing

Advances in Pharmacology, Volume 68 # 2013 Elsevier Inc. 37

aromatic L-amino acid decarboxylase (AADC), lacking the DA membrane transporter

Hökfelt, Johansson, & Goldstein, 1984; Jaeger & Teitelman, 1992;

Aromatic L-amino acid

HO CH2 CH2 NH2

Figure 4.1 Dopamine (DA) synthesis from L-tyrosine. L-DOPA, L-3,4-

Figure 4.2 Schematic representation of DA turnover in dopaminergic (DA-ergic) neu-

Figure 4.3 Schematic representation of location of major clusters of DA-ergic neurons

Neurons containing catecholamines including DA have been first iden-

method (Björklund & Lindvall, 1984), autoradiography following adminis-

2. NEURONS PARTLY EXPRESSING DA-ERGIC

Table 4.1 Transient populations of monoenzymatic TH neurons (cell bodies or fibers)

2.2. Distribution of “monoenzymatic” neurons in the brain

Figure 4.4 Tyrosine hydroxylase (TH)-immunoreactive neurons (A) and aromatic L-

According to the double-immunolabeling study, the total number of

number of the monoenzymatic TH neurons and bienzymatic neurons in

Density of fibers/unity of ME area 10 ∗

In contrast to the monoenzymatic TH axons, the concentration of the

2.2.2 Suprachiasmatic nucleus

monoenzymatic TH neurons. The former neuron population is maintained in

postnatal weeks and then almost disappeared by prepuberty, simultaneously

2.2.3 Magnocellular nuclei

3. NEURONS PARTLY EXPRESSING DA-ERGIC

overlapping of these populations (Okamura, Kitahama, Nagatsu, et al.,

3.2. Distribution of monoenzymatic neurons in the brain

particular attention that is explained by the crucial role of these brain

Calas, Makarenko, et al., 2002). Moreover, according to the double-

in the preceding text that the population of monoenzymatic neurons at least

Figure 4.9 Double-immunolabeling at the electron microscopic level of TH (electron-

Special attention was paid to topographic relation of bienzymatic and

Figure 4.10 Axosomatic specialized-like contact (arrowhead) between monoenzymatic

specialized-like junctions were observed with confocal microscopy (Ershov,

contacts obtained in confocal microscope, were further proved with elec-

3.2.2 Anterior hypothalamus

Figure 4.11 Schematic representation of the distribution of TH-immunoreactive neu-

et al., 1998). Besides, occasional monoenzymatic TH neurons were detected

Figure 4.12 Schematic representation of the distribution of TH-immunoreactive neu-

The striatal TH-immunoreactive neurons in monkeys are usually round

4. FUNCTIONING OF MONOENZYMATIC NEURONS

4.1. Monoenzymatic neurons expressing TH

vasopressinergic neurons of the human supraoptic nucleus was recently shown

as it was suggested by the authors (Cossette, Lecomte, et al., 2005; Cossette,

(Melnikova et al., 1999) containing numerous monoenzymatic TH neurons

of neurotransmitters including g-aminobutyric acid, neurotensin, somato-

4.2. Monoenzymatic neurons expressing AADC

4.3. Monoenzymatic neurons expressing either TH or aromatic

separately, but only according to our methodology; the neurons expressing

the DA and, hence, 6-hydroxydopamine uptake in this peculiar brain region

4.3.2 Cooperative synthesis of DA in adulthood

and serotoninergic neurons located in other brain regions, mostly in the

membrane transporter in monoenzymatic AADC neurons of the MBH of

resulted in a drop of the DA concentration in the incubation medium after

Figure 4.16 Schematic representation of cooperative synthesis of DA by

Neuron functioning and functional significance in general are believed to

Prolactin in plasma (ng/ml)