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Copyright © 2001 by Institute of Pharmacology
Polish Academy of Sciences
Polish Journal of Pharmacology
Pol. J. Pharmacol., 2001, 53, 681–684
ISSN 1230-6002
PRELIMINARY COMMUNICATION
SEARCH FOR THE PRESENCE OF GLUCOCORTICOID
RECEPTORS IN DOPAMINERGIC NEURONS OF RAT
VENTRAL TEGMENAL AREA AND SUBSTANTIA NIGRA
Anna Czyrak, Agnieszka Chocyk
Laboratory of Pharmacology and Brain Biostructure, Institute of Pharmacology, Polish Academy of Sciences,
Smêtna 12, PL 31-343 Kraków, Poland
Search for the presence of glucocorticoid receptors in dopaminergic neurons of rat
ventral tegmenal area and substantia nigra. A. CZYRAK, A. CHOCYK. Pol. J. Pharmacol., 2001, 53, 681–684.
Using non-fluorescent immunocytochemical double-labelling procedure and specific
antibodies visualizing GR (glucocorticoid receptors) and TH (tyrosine hydroxylase) we
have been looking for the co-localization of both antigens in neurons of the rat ventral
tegmental area and adjacent substantia nigra. This experimental direction has been inspired by the available data showing that alterations in the level of circulating glucocorticosteroids have distinct effects on the intensity of dopaminergic neurotransmission. Thus,
it was of interest to find the anatomical background for the above interaction. It has been
found that the rat ventral tegmental area and substantia nigra possess a relatively moderate number of cells with active GR, i.e. receptors which are condensed in the nuclei. Further, we found that dopaminergic neurons (TH-positive) of the ventral tegmental area and
substantia nigra were not immunopositive for GR. This observation was in the sharp contrast to the results from the locus coeruleus, where the co-localization of GR with TH was
a general rule. Above anatomical data indicate that glucocorticoid receptors influence the
dopaminergic neurotransmission by an indirect mechanism, which possibly involves intermittent neurotransmitter.
Key words: double-labelling, glucocorticoid receptors, locus coeruleus, substantia nigra, tyrosine hydroxylase, ventral tegmental area
correspondence; e-mail: [email protected]
A. Czyrak, A. Chocyk
Glucocorticoids operating via their receptors
which are hormone-activated transcription factors
exert remarkable influence on various neurotransmitters in the central nervous system [4]. Above effects involve alterations of neurotransmitters synthesis and release, changes in synthesis of specific
proteins (enzymes and receptors), apart from alterations of the anatomical shape of certain populations of hippocampal neurons [1, 2, 3, 4, 7]. In this
respect, several arguments indicate that glucocorticoids, or specifically alterations in their circulating
concentrations, exceeding their physiological levels,
have profound effects on the dopaminergic neurotransmission. For example, it has been observed that
corticosterone, operating via GR alters the turnover
rate and release of dopamine, evokes changes in the
density of dopaminergic receptors of D1 subtype,
with the subsequent alteration in their mRNA level
[1, 3, 7]. It has also been found that corticosterone
alters the responsiveness of animals to addictive
psychostimulants, which are acting via dopaminergic systems [8]. In spite of the above neurochemical and behavioral arguments for the interaction
between glucocorticosteroids and dopaminergic
neurotransmission, the cellular mechanism of this
interaction is not fully understood.
Therefore, in the present study we investigated
whether corticosterone receptors of GR subtype are
present in the dopaminergic cell bodies of the rat
ventral tegmental area and adjacent substantia nigra, the two major groups of dopaminergic neurons
forming mesolimbic, mesocortial and mesostriatal
dopaminergic systems. This experimental direction
was based on the assumption that presence of GR,
which operates as transcripctional factors [4, 5] in
dopaminergic neurons may provide anatomical evidence that corticosterone via GR may directly modify the function of dopaminergic cells and subsequently the intensity of dopaminergic neurotransmission.
All the experiments were carried out on male
Wistar rats (200–250 g) housed in groups (6 animals per cage) under an artificial light/dark cycle
(12/12 hours, light on at 07:00. Control (vehicle-treated) and corticosterone-injected (10 mg/kg sc)
rats were deeply anesthetized (1 h after the treatment) with sodium pentobarbital (100 mg/kg) and
were transcardially perfused with saline (NaCl,
0.9%) followed by an ice cold 4% paraformaldehyde in a 0.1 M PBS (phosphate buffered saline).
Following a 4-hour postfixation period (4% para-
682
A
c
VTA
SN
r
B
C
D
Fig. 1.
Photomicrographs of the rat ventral tegmental area and
substantia nigra stained for the presence of TH (brown) and
)
GR (black), (
) low power magnification (objective 4x) with
marked areas presented in high power magnification photomicrographs (objective 40x) from substantia nigra pars compacta
*
(
+
), substantia nigra pars reticulata (
,
area (
) and ventral tegmental
); arrows – neurons positive for the presence of TH,
arrowheads – neuronal nuclei GR-immunopositive. Abbreviations: VTA – ventral tegmental area; SN – substantia nigra, c –
pars compacta, r – pars reticulata
Pol. J. Pharmacol., 2001, 53, 681–684
GLUCOCORTICOID RECEPTORS AND DOPAMINERGIC NEURONS
)
Fig. 2. Photomicrographs of the rat locus coeruleus stained for the presence of TH (brown) and GR (black), (
) low power magnifica-
*
tion (objective 10x) with marked area presented in (objective 40x) high power magnification photomicrograph (
), TH-positive lo-
cus coeruleus neurons (cytoplasm) are also immunopositive for the presence of GR receptors (nucleus). Abbreviations: LC – locus
coeruleus, 4v – fourth ventricle
formaldehyde in 0.1 M PBS, as a fixative), 50 mm
thick sections were cut at the level of substantia
nigra, ventral tegmental area and locus coeruleus,
using a vibratome (Technical Products International, USA). The sections were rinsed extensively in
0.05 M TBS (Tris buffered saline) and then incubated for 1 h in blocking buffer containing 3% normal goat serum (Vector Lab., USA), 0.1% bovine
serum albumin in TBS. Then the sections were incubated with primary polyclonal rabbit antibody
(Affinity Bioreagents INC., USA) against 346–367
amino acids from N-terminus of human GR, diluted at 1:1000 in the blocking buffer (48 h at 4°C).
After incubation and several washes, the sections
were incubated (1 h) with biotinylated, secondary
goat anti-rabbit antibody (Vector Lab.). The final
incubation of the sections with a solution of the
avidin-biotin horseradish peroxidase complex (Vector Lab.) was preceded by the 30 min incubation of
sections in 0.6% H2O2. The reaction was visualized
by nickel-glucose oxidase-diaminobenzidine method, giving the black color of staining which was
in a good contrast to the brown deposits of TH (see
below). After washing in 0.01 M PBS, free-floating
sections were incubated for 60 min in a blocking
buffer containing 2% normal horse serum, 0.2%
ISSN 1230-6002
Triton X-100, and 0.1% bovine serum albumin in
0.01 M PBS. Then the sections were incubated
(48 h, 4°C, continuos agitation) with primary monoclonal antibody, recognizing the TH (Chemicon,
USA) diluted at 1:5000 in blocking buffer. After
rinsing, the sections were incubated for 1 h with
biotinylated anti-mouse, IgG (rat adsorbed) antibody (Vector Lab.) diluted in the blocking buffer.
The reaction was visualized by incubation with
avidin-biotin horseradish peroxidase complex (Vectastain Elite ABC Kit, Vector Lab.) and developed
by diaminobenzidine-hydrogen peroxide reaction,
which produced the brown staining of immunopositive material.
It has been found that GR are present in the
ventral tegmental area and adjacent substantia nigra (both, pars compacata and pars reticulata) as
well as in the locus coeruleus. The GR protein was
present in the cell nuclei, which indicates that our
antibody visualized the active form of GR, i.e. receptors which are activated by corticosterone and
translocated to the nucleus [4, 5]. We observed a similar degree of nuclear condensation of immunopositive material in naive and corticosterone-treated
animals which suggests that in the studied brain regions, the vast majority of GR, are in the active
683
A. Czyrak, A. Chocyk
form already in physiological conditions. The size
of GR-immunopositive nuclei varied from relatively large ones in the locus coeruleus to large
ones and moderately small in the substantia nigra
and ventral tegmental area. Size of stained nuclei
may indicate that in the locus coeruleus, GR are
present mainly in neurons, whereas in the ventral
tegmental area and substantia nigra they are present
in neurons and glia cells in comparable amounts.
The monoclonal antibody visualizing TH showed
a typical pattern of distribution of this enzyme in
all brain regions tested [5]. The double-labelling
procedures revealed that TH-positive neurons of
both ventral tegmental area and substantia nigra did
not show GR-immunopositive material. Above data
are in sharp contrast to the data collected from the
locus coeruleus, which has been used as a positive
control, where we observed a clear co-localization
of GR with TH.
In conclusion, the obtained results indicate that
dopaminergic neurons of the rat ventral tegmental
area and substantia nigra, i.e. neurons which contain enzyme TH, are not immunopositive for GR,
although these receptors are present in above brain
regions both in neurons and also glia cells. The
present anatomical data indicate that the impact of
glucocorticoids on dopaminergic neurotransmission is not a direct one, but possibly involves the
intermittent neurotransmitter that tunes the activity
of dopaminergic neurons. It will be of importance
to analyze which neurotransmitter is involved in
the above effects. Among the possible candidates,
glutamatergic system should be taken into consideration, which innervates the ventral tegmental
area and substantia nigra, originating in the cortex
(among other structures). Interestingly, we observed
that almost all pyramidal neurons, i.e. glutamatergic neurons in the cortex, contain GR immunoreactivity (our unpublished observations). Moreover,
alterations in the glutamate outflow in the ventral
tegmental area have been linked with the phenomenon of sensitization to psychostimulants [6]. Finally, it has been also found that release of glutamate
is controlled by dopamine D1 receptors which are
localized on the glutamatergic terminals originating
in the cortex [9, 10]. Density of D1 dopamine receptors is controlled by glucocorticoids, what was
684
shown by our previous experiments [3]. Additional
important information obtained in the present study
is that the appearance of GRE sequences (glucocorticoid response element) in the promoter of TH
gene does not indicate that GR may directly influence TH expression in every single neuron. It is apparent from our study that direct interaction at the
level of single neuron would depend on the type of
neurotransmitter and brain structure.
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Received: October 25, 2001; in revised form: November
20, 2001.
Pol. J. Pharmacol., 2001, 53, 681–684