Molecular basis for catecholaminergic
Jan Grimm*†‡, Anne Mueller§, Franz Hefti*‡, and Arnon Rosenthal*‡
*Rinat Neuroscience, 3155 Porter Drive, Palo Alto, CA 94304; and§Department of Microbiology and Immunology, Stanford University School of Medicine,
Stanford, CA 94305-5402
Communicated by Cornelia I. Bargmann, University of California, San Francisco, July 23, 2004 (received for review May 13, 2004)
Catecholaminergic neurons control diverse cognitive, motor, and en-
docrine functions and are associated with multiple psychiatric and
neurodegenerative disorders. We present global gene-expression
profiles that define the four major classes of dopaminergic (DA)
and noradrenergic neurons in the brain. Hypothalamic DA neurons
and noradrenergic neurons in the locus coeruleus display distinct
group-specific signatures of transporters, channels, transcription,
plasticity, axon-guidance, and survival factors. In contrast, the
the ventral tegmental area are closely related with <1% of dif-
ferentially expressed genes. Transcripts implicated in neural plas-
ticity and survival are enriched in ventral tegmental area neurons,
consistent with their role in schizophrenia and addiction and their
decreased vulnerability in Parkinson’s disease. The molecular pro-
files presented provide a basis for understanding the common and
population-specific properties of catecholaminergic neurons and
will facilitate the development of selective drugs.
nized in anatomically discrete groups and constitute ?1 of 107
cells in the vertebrate CNS (1, 2). The most prominent groups
of DA neurons reside in the substantia nigra (SN; A9 cell group;
?10,000 neurons in the rat) and the ventral tegmental area
(VTA; A10; ?25,000 neurons) of the midbrain. The SN neurons
provide the nigrostriatal ascending inputs to the telencephalon
and comprise a key component of the extrapyramidal motor
system controlling postural reflexes and initiation of movement.
DA neurons in the adjacent VTA give rise to mesocortical and
mesolimbic pathways that are implicated in control of emotional
balance, reward-associated behavior, attention, and memory.
Additional groups of DA neurons reside in the medial zona
incerta of the hypothalamus (A13; ?900 neurons) and partici-
pate in the regulation of endocrine functions. The largest
collection of NA neurons resides in the pontine locus coeruleus
(LC; ?1,500 neurons). These neurons form a modulatory pro-
jection system to most CNS areas and contribute to the regula-
tion of emotional status, sensory perception, arousal, sleep–
wake patterns, and most autonomic functions.
Consistent with their varied functions, the CA neurons are
associated with multiple neurodegenerative, psychiatric, and
endocrine disorders. Selective degeneration of DA neurons in
the SN but not in the VTA or the hypothalamus leads to
Parkinson’s disease (PD) (3–7), whereas abnormal function of
the VTA DA neurons has been linked to schizophrenia, atten-
tion deficit, addiction, and hyperactivity disorders (8–11). In
addition, dysfunction of hypothalamic DA neurons can cause
hyperprolactinemia, an endocrine disorder of the reproductive
system (12), whereas changes in the activity of the NA system
have been linked to depression as well as sleep disorders (13, 14).
The drugs currently available for therapy reflect the critical
role of CA systems in disease states. Compounds substituting for
the diminished levels of DA in SN neurons are palliative for PD
(15–17), whereas drugs blocking the function of VTA neurons
are the mainstay therapy for schizophrenia (18). DA drugs are
also used to control hyperprolactinemia, whereas antidepressant
atecholaminergic (CA) neurons producing the neurotrans-
mitters dopamine (DA) and noradrenalin (NA) are orga-
drugs (13) and also drugs of abuse, such as amphetamines and
cocaine, act on transporter systems of CA neurons (8, 9).
Because these drugs do not distinguish between classes of CA
neurons, the desired effects in one condition become the adverse
effects in another disease. For example, PD-like extrapyramidal
motor disturbances are common in schizophrenia drug therapy,
whereas hallucinations and paranoia are common side effects of
DA drug therapy for PD and hyperprolactinemia. Here, we
report the genome-wide expression profiles of four major sub-
populations of adult CA neurons. This study provides a foun-
dation for mechanistic understanding of the development, ste-
reotypic positions, control of innervation targets, function, and
disease vulnerability of these neuronal classes and for the
identification of selective drug targets.
Materials and Methods
Tissue Preparation and Immunohistochemistry. Adult (7- to
9-month-old) female Sprague–Dawley rats were anesthetized
and killed by decapitation. The brains were rapidly dissected and
immediately frozen on dry ice. We mounted 12-?m cryosections
on polyethylene-naphthalene membrane slides pretreated with
0.1% poly-L-lysine for 5 min, followed by 30 min of UV
for 30 s, followed by acetone for 3 s, and air-dried. After
rehydration in PBS (pH 7.0) for 5 s, the sections were stained for
2 min in PBS (pH 7.0) containing 100 ?g?ml anti-tyrosine
hydroxylase (TH) Ab (clone TH-16; Sigma) that had been
labeled with the Alexa Fluor 488 mAb-labeling kit (Molecular
Probes) according to the manufacturer’s instructions. Rehydra-
tion and staining were performed in the presence of 1 unit??l
RNase inhibitor (Roche). The sections were washed twice in
PBS for 5 s; dehydrated for 30 s in 75%, 95%, and 100% ethanol,
respectively; and air-dried at room temperature.
Laser Microdissection, RNA Isolation, and Amplification. Single neu-
rons were isolated from immunostained cryosections by using a
PALM Robot–Microbeam system (PALM Microlaser Technol-
ogy, Bernried, Germany). To facilitate detection of fluorescent
neurons, a drop of 100% ethanol was applied to the section
during cell selection. The sections were allowed to air dry, and
neurons were dissected and catapulted into 30 ?l of lysis buffer.
Total RNA from 200 pooled neurons was isolated by using the
PicoPure kit (Arcturus, Mountain View, CA), and contaminat-
ing genomic DNA was removed during the isolation by an
on-column DNase digestion step. The common reference RNA
was generated from three whole brains of age-matched female
rats. RNA was isolated by using RNA-Bee (Tel-Test, Friends-
wood, TX), followed by DNase digestion with the DNAfree kit
Abbreviations: CA, catecholamine?catecholaminergic; DA, dopamine?dopaminergic; LC,
locus coeruleus; NA, noradrenalin?noradrenergic; PD, Parkinson’s disease; SN, substantia
nigra; VTA, ventral tegmental area; TH, tyrosine hydroxylase; DBH, DA-?-hydroxylase.
†To whom correspondence should be addressed. E-mail: email@example.com.
‡A.R., F.H., and J.G. have filed a patent application covering the methodology and several
genes described in this article.
© 2004 by The National Academy of Sciences of the USA
September 21, 2004 ?
vol. 101 ?
no. 38 ?
regulates emotional, cognitive, and sleep–wake functions, ex-
pressed the highest number of specific genes. In contrast,
hypothalamic A13 neurons, which have simpler projections and
control mainly endocrine functions, displayed the lowest tran-
Despite the high similarity of the transcriptomes in SN and
VTA neurons, we were able to identify a number of subpopu-
lation-specific genes. Among the gene transcripts enriched in the
VTA, several encoded proteins that are implicated in synaptic
plasticity. These factors may contribute to the long-term synaptic
plasticity elicited by psychostimulants, leading to drug addiction
(43). A critical role of PLC?1 in the regulation of long-term
adaptations to drugs has recently been demonstrated by over-
expression experiments in the VTA (44). Likewise, the expres-
sion of the learning- and plasticity-associated immediate-early
gene Zif268 is induced in VTA neurons upon drug-conditioned
stimulation and decreases during prolonged withdrawal (45, 46).
VTA neurons were also enriched for several factors that are
involved in axonal pathfinding and neuronal migration (neuro-
pilin-1, slit-2, and ephrin B3). During development, SN neurons
target mainly the dorsolateral striatum, whereas VTA neurons
mainly innervate the ventromedial striatum. The molecular
signals that regulate the development of these pathways have
been characterized only partially (47). The differential expres-
sion of multiple members of the ephrin?Eph and slit?robo
families identified here could have important functions in the
maintenance and stability of neuronal networks, in adult plas-
ticity and remodeling, and possibly in schizophrenia, a disease
that is most likely linked to abnormal development of cortical
areas innervated by VTA neurons (48).
One of our goals was to identify genes that may influence the
selective vulnerability of CA neurons in PD. The SN is most
susceptible to PD pathology, whereas the adjacent VTA DA
neurons are less vulnerable and hypothalamic DA neurons are
spared (3–6, 49). The same selective vulnerability of DA neuron
subpopulations has been observed in rodent and primate models
of PD (7, 50–52). The sparing of VTA neurons could be
mediated by selective expression of neuroprotective factors,
including neurotrophic factors (BMP-2, PACAP, and ANP),
detoxifying enzymes (EC-SOD, lipoprotein lipase, and UDP-
glucuronosyltransferase), the antiapoptotic factor PARM-1, and
decreased levels of the proapoptotic PKC-?. We also observed
selective high expression of ?-synuclein in neurons of the SN and
in LC NA that degenerate in PD, which may modify the toxic
effects of the widely expressed ?-synuclein protein. Likewise,
selective expression of the Zn2?transporter by the SN and VTA
may play a role in the pathophysiology of PD. Low concentra-
tions of Zn2?can exert a cell-protective effect; however, excess
of Zn2?is neurotoxic and has been shown to promote degen-
eration of midbrain DA neurons (53).
In summary, the molecular signatures of the major classes of
CA neurons can advance our understanding of the characteristic
features and functions of these neurons and facilitate the dis-
covery of subgroup-selective drug targets.
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www.pnas.org?cgi?doi?10.1073?pnas.0405340101Grimm et al.