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The Journal of Neuroscience, December 15, 1998, 18(24):10700-10708
Differential Regulation of Neurotrophin and trk
Receptor mRNAs in Catecholaminergic Nuclei during Chronic Opiate
Treatment and Withdrawal
Suzanne
Numan1,
Sarah
B.
Lane-Ladd1, 2,
Lixin
Zhang4,
Kerstin H.
Lundgren4,
David S.
Russell1, 3,
Kim B.
Seroogy4, and
Eric J.
Nestler1, 2
Laboratory of Molecular Psychiatry, Departments of
1 Psychiatry, 2 Neurobiology, and
3 Neurology, Yale University School of Medicine and
Connecticut Mental Health Center, New Haven, Connecticut 06508, and
4 Department of Anatomy and Neurobiology, University of
Kentucky College of Medicine, Lexington, Kentucky 40536
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ABSTRACT |
The neurotrophins brain-derived neurotrophic factor (BDNF) and
neurotrophin-3 (NT-3) and their receptors trkB and
trkC, respectively, are expressed in the locus coeruleus
(LC) and ventral tegmental area (VTA), brain regions known to be
involved in opiate addiction. Previously, administration of exogenous
neurotrophins has been shown to oppose effects of chronic morphine
treatment on LC and VTA neurons. However, the response of endogenous
neurotrophins in LC and VTA to opiate treatment is unknown. In this
study, BDNF, NT-3, trkB, and trkC mRNAs
were analyzed in these regions after chronic morphine treatment and
during antagonist precipitated withdrawal. Although chronic morphine
exposure resulted in only modest increases in BDNF and NT-3 mRNA
expression in LC, precipitated withdrawal led to a marked, rapid, and
prolonged increase in BDNF mRNA and a delayed decrease in NT-3 mRNA.
Levels of trkB and trkC mRNAs, which were
unchanged by chronic morphine treatment, were elevated in LC at 2 and 6 hr of withdrawal. By 20 hr, trkB mRNA levels in LC had
returned to control, whereas trkC mRNA levels fell below
control values. In contrast to the substantial alterations observed in
LC, there was no regulation of the neurotrophins or trk
mRNAs within the VTA during chronic opiate treatment or withdrawal, with the exception of an increase in trkB mRNA at 6 hr
of withdrawal. These results suggest that neurotrophins and their
receptors per se may be involved in opiate-induced plasticity of the
LC, whereas other mechanisms would appear to be involved in the VTA.
Key words:
locus coeruleus; ventral tegmental area; opiates; neurotrophins; neural plasticity; morphine
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INTRODUCTION |
The locus coeruleus (LC), the major
noradrenergic cell group in the brain (for review, see Aston-Jones et
al., 1995 ), has been implicated in opiate physical dependence and
withdrawal (Nestler and Aghajanian, 1997). Acutely, opiates inhibit LC
neuronal activity (Korf et al., 1974; Bird and Kuhar, 1977; Valentino
and Wehby, 1988). In response to continued opiate treatment, several
long-term biochemical alterations occur in LC, including increases in
tyrosine hydroxylase, adenylyl cyclase, and protein kinase A (Duman et al., 1988; Nestler and Tallman, 1988; Guitart et al., 1990; Nestler et
al., 1993). In addition, tolerance develops such that LC neuronal firing rates return toward control levels, and the neurons show dependence such that abrupt opiate removal causes marked increases in
LC neuronal activity (Aghajanian, 1978; Valentino and Wehby, 1989;
Rasmussen et al., 1990). This increase in LC activity appears both necessary and sufficient to induce many opiate withdrawal behaviors (Grant et al., 1988; Maldonado and Koob, 1993; Nestler and
Aghajanian, 1997).
Another catecholaminergic nucleus implicated in opiate addiction is the
ventral tegmental area (VTA) (Wise, 1996; Koob and Le Moal, 1997; Koob
and Nestler, 1997). This nucleus, which contains dopaminergic neurons
that project to the nucleus accumbens and other forebrain regions (for
review, see Lindvall and Björklund, 1983), is thought to be
involved in rewarding aspects of opiate administration. Like the LC,
the VTA undergoes long-term biochemical changes after chronic
opiate exposure, including increases in tyrosine hydroxylase and
certain glutamate receptor subunits and decreases in neurofilaments
(Nestler et al., 1993, 1996). Although opiates acutely activate VTA
dopamine neurons by inhibiting inhibitory interneurons (Johnson and
North, 1992), the chronic electrophysiological effects of opiates on
these cells have only recently been investigated (Bonci and Williams,
1997).
Recent evidence suggests that neurotrophins, in addition to their well
known roles in neuronal development and survival, may be involved in
maintenance of neuronal phenotype, neuronal plasticity, and
neuroprotection for adult neurons (Gall, 1993; Korsching, 1993; Lindsay
et al., 1994). Brain-derived neurotrophic factor (BDNF) and
neurotrophin-3 (NT-3) are two neurotrophins that increase survival of
LC neurons (Friedman et al., 1993; Sklair-Tavron and Nestler, 1995) and
ventral midbrain dopamine neurons (Hyman et al., 1991, 1994; Spenger et
al., 1995) in vitro. Both BDNF and NT-3 mRNAs and their
cognate receptor mRNAs trkB and trkC,
respectively, are expressed in adult LC (Seroogy, 1994; Smith et al.,
1995; Zhang et al., 1998) and VTA (Gall et al., 1992; Seroogy and Gall, 1993; Altar et al., 1994; Seroogy et al., 1994; Numan and Seroogy, 1998). Furthermore, neurotrophin mRNA expression can be
regulated in these nuclei by specific pharmacological treatments (Numan and Seroogy, 1994; Seroogy, 1994; Hung and Lee, 1996).
Recent studies suggest a role for neurotrophins in opiate addiction.
Administration of exogenous BDNF or NT-3 opposes effects of chronic
opiate treatment on LC neurons in vitro (Sklair-Tavron and
Nestler, 1995) and on VTA neurons in vivo (Berhow et al., 1995; Sklair-Tavron et al., 1996). However, the response of endogenous neurotrophin systems in the LC and VTA to opiate treatment is unknown.
In the present study, expression of mRNAs for BDNF and NT-3 and for
their high-affinity receptors trkB and trkC,
respectively, was examined in the LC and VTA after chronic morphine
treatment and during opiate withdrawal to study possible plasticity of
these neurotrophin systems in the context of opiate addiction.
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MATERIALS AND METHODS |
Animals and treatments
Adult male Sprague Dawley rats (150-200 gm; n = 32; CAMM, Wayne, NJ) were used for this study. The opiate treatment
paradigms used in these experiments followed protocols described
previously by Rasmussen and colleagues (1990).
Chronic morphine. For 5 d, once a day, rats
(n = 4) were lightly anesthetized with fluothane
and implanted with a morphine pellet (75 mg of morphine base; National
Institute on Drug Abuse) subcutaneously. Control rats
(n = 4) were lightly anesthetized but not implanted
with pellets. On day six all rats were decapitated.
Morphine withdrawal (short-term survival). Rats were treated
as above; however, on day six, sham-treated rats (n = 4) were injected with 0.9% saline subcutaneously and decapitated 2 hr later. Morphine-treated rats were injected with naltrexone (100 mg/kg
in 0.9% saline, s.c.), housed individually, and decapitated 2 hr
(n = 4) or 6 hr (n = 4) after the injection.
Morphine withdrawal (long-term survival). Rats were treated
as in the chronic morphine paradigm; however, on day six, sham-treated rats (n = 4) were killed by rapid decapitation.
Morphine-treated rats had their last two implanted morphine pellets
removed under light fluothane anesthesia and then were injected with
naltrexone (100 mg/kg in 0.9% saline, s.c.) and housed separately.
After 6 hr of withdrawal, rats received another injection of
naltrexone. Some of the rats (n = 4) were decapitated
after 20 hr of withdrawal. Finally, the remaining group
(n = 4) received a third injection of naltrexone after
24 hr of withdrawal and were decapitated at 70 hr. For the long
withdrawal periods, rats were supplied with mashed food and water in
the cage with them as well as with a local heat source.
Tissue collection
After decapitation, brains were quickly removed and frozen with
dry ice. Coronal sections (10 µm) throughout the rostrocaudal extent
of the LC and the VTA were cut in a cryostat, thaw-mounted onto
Superfrost Plus (Fisher Scientific, Pittsburgh, PA) glass slides, and
stored at  20°C until hybridization.
In situ hybridization
Coronal sections through the rostrocaudal extent of the LC and
VTA were processed for the in situ hybridization
localization of BDNF, NT-3, trkB, and trkC mRNAs
by using 35S-labeled cRNA probes as described previously
(Seroogy et al., 1994; Numan and Seroogy, 1997; Seroogy and Herman,
1997). These nuclei were identified by standard anatomical landmarks
(Paxinos and Watson, 1986). Briefly, the slide-mounted sections were
brought to room temperature and then placed in 4% paraformaldehyde for 10 min. This was followed by washes in 0.1 M phosphate
buffer (PB), 0.1 M PB/0.2% glycine, and 0.25% acetic
anhydride in 0.1 M triethanolamine. The sections were then
dehydrated with increasing concentrations of ethanol, delipidated in
chloroform, and air-dried. Sections were hybridized overnight at 60°C
in a hybridization mixture consisting of 10% dextran sulfate, 50%
formamide, 1× Denhardt's solution, 0.15 mg/ml yeast tRNA, 40 mM dithiothreitol, 0.33 mg/ml denatured salmon sperm DNA, 1 mM EDTA, 20 mM Tris-HCl, and the 35S-labeled cRNA probe at a concentration of 1.0 × 106 cpm/50 µl per slide. Sense and antisense cRNA
probes were prepared by in vitro transcription using
linearized DNA constructs in the presence of RNA polymerase (T3,
T7, or SP6) and 35S-UTP (New England Nuclear, Boston, MA).
BDNF and NT-3 cDNA constructs (kindly supplied by J. Lauterborn and C. Gall, University of California at Irvine) resulted in antisense
transcripts that were 540 and 550 bases long, respectively. The cDNA
constructs for trkB and trkC (generous gifts from
D. McKinnon, State University of New York at Stony Brook) resulted in
antisense RNA transcripts that were 196 and 300 bases long,
respectively. The trkB cRNA probe used in this study
detected only the kinase-specific, full-length catalytic form of the
receptor mRNA (Sternini et al., 1996). In contrast, the probe used to
detect trkC mRNA recognized mRNA transcripts for both the
full-length catalytic and truncated noncatalytic forms of the receptor
(Dixon and McKinnon, 1994; Albers et al., 1996).
For treatment after hybridization, sections were washed several
times in 4× SSC (1× SSC = 0.15 M sodium chloride,
0.015 M sodium citrate, pH 7.0) containing 10 mM sodium thiosulfate, at 37°C. The sections were then
incubated in ribonuclease A (0.05 mg/ml) for 30 min at 45°C. This was
followed by several washes in decreasing concentrations of SSC (2, 0.5, and 0.1×) at 37°C. All but the final wash also contained 10 mM sodium thiosulfate. The sections were briefly rinsed in
dH20, dipped in 95% ethanol, and finally air-dried. To
generate film autoradiograms the sections were exposed to  -Max
Hyperfilm (Amersham, Arlington Heights, IL) for 7-15, 7-18, 14-22,
and 21-25 d for trkC, trkB, BDNF, and NT-3
mRNAs, respectively. It should be noted that the variability in film
exposure for a single probe comes from the different experiments (chronic morphine, 2 and 6 hr morphine withdrawal, or 20 and 70 hr
withdrawal in LC or VTA) and not from within a single experiment. Therefore, for each probe all of the groups for one experiment (e.g.,
control, 2 and 6 hr withdrawal in LC) were processed at the same time
and were on film for the same amount of time. As controls for
specificity, some sections were pretreated with ribonuclease A (0.05 mg/ml) for 30 min at 45°C before hybridization with the 35S-labeled cRNA probes. Some sections were also hybridized
with BDNF, NT-3, trkB, or trkC sense strand
35S-labeled riboprobes. No specific labeling was observed
under any of these control conditions.
Film autoradiograms were analyzed by densitometry using NIH Image
software to compare densities of hybridization of each probe in the LC
and VTA between control rats and treated rats. From each animal, at
least six measurements of hybridization density, also referred to as
gray level, were taken for each probe from both LC and VTA. A paired
background measurement was subtracted from each gray level, leading to
a corrected gray level. Once a mean corrected gray level for LC and VTA
was attained for each animal, statistical analysis was performed.
Statistical analysis included Student's unpaired t test and
ANOVA followed by Student Newman-Keuls post hoc analyses
where appropriate. The NIH image software was also used to acquire
images of representative sections from film autoradiograms.
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RESULTS |
Locus coeruleus
Chronic morphine
Coronal sections throughout the LC of control and morphine-treated
rats were processed for the in situ hybridization
localization of BDNF, NT-3, trkB, and trkC mRNAs.
Densitometric analysis of film autoradiograms revealed a small but
significant increase (+10 ± 1.8%) in BDNF mRNA expression in the
LC after chronic morphine treatment as compared with control rats
(p < 0.05) (Fig.
1A, Table 1). Similarly, chronic morphine
administration induced a slight increase (+8 ± 1.9%) in NT-3
mRNA expression in this brain region (p < 0.05)
(Fig. 1B, Table 1). In contrast to the small
increases in neurotrophin mRNA expression in the LC after chronic
opiate treatment, trkB and trkC cRNA
hybridization densities were not significantly altered
(p > 0.05) (Fig. 1C,D, Table 1).
However, it should be noted that there did appear to be a trend toward a decrease (9 ± 2.1%) in trkC mRNA levels in
morphine-treated versus control rats (p = 0.062)
(Fig. 1D, Table 1).
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Figure 1.
Analysis of BDNF (A), NT-3
(B), trkB
(C), and trkC
(D) mRNA expression in LC after chronic opiate
treatment. Note the significant increase in BDNF
(A) and NT-3 (B) mRNA
levels (asterisks) in LC after chronic opiate exposure.
Although there was a trend toward a decrease in trkC
mRNA levels (D; p = 0.062), no
significant alterations in trkB
(C) or trkC
(D) mRNA expression were observed in LC with
chronic morphine treatment. Results are expressed as a percentage of
control values. Values are the mean ± SEM.
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Morphine withdrawal
Visual inspection of film autoradiograms revealed a substantial
increase in BDNF mRNA levels in the LC after 2 hr of morphine withdrawal as compared with control animals (Fig.
2A). There also appeared to be a further elevation in the BDNF cRNA hybridization density after 6 hr of withdrawal (Fig. 2A).
Densitometric analysis of film autoradiograms confirmed that there were
significant alterations in BDNF mRNA levels during precipitated opiate
withdrawal [F(2,8) = 223.451; p < 0.001]. Post hoc analyses revealed that BDNF mRNA levels
were significantly raised at 2 hr (+100 ± 5%) and 6 hr (+147 ± 1%) of morphine withdrawal as compared with control
levels (p < 0.05) (Fig. 2C, Table
1). Furthermore, BDNF mRNA levels in the LC were higher at 6 hr of
morphine withdrawal than at 2 hr (p < 0.05)
(Fig. 2C, Table 1).
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Figure 2.
Effect of precipitated opiate withdrawal on BDNF
mRNA expression in LC. A, Film autoradiograms
demonstrating BDNF cRNA hybridization in LC of control rats and rats
exposed to 2 or 6 hr of opiate withdrawal. B, Film
autoradiograms of BDNF mRNA expression in LC in control rats and rats
exposed to 20 or 70 hr of opiate withdrawal. C,
Densitometric analysis of film autoradiograms reveals robust increases
in BDNF mRNA levels at 2, 6, 20, and 70 hr of opiate withdrawal as
compared with control (p < 0.05). Results
are expressed as a percentage of control values. Values are the
mean ± SEM (SEM for 2 and 6 hr control group = ±6; SEM for
20 and 70 hr control group = ±6.5.) *, Significantly
increased from control. ** Significantly increased from control
and 2 hr. Significantly different from control and 20 hr. Scale bar, 500 µm.
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To study the duration of BDNF mRNA induction during withdrawal, another
set of animals was processed for the in situ hybridization localization of the neurotrophins and their receptors in the LC after
longer withdrawal periods. As with the earlier time points, robust
increases in BDNF mRNA levels occurred after longer periods of
withdrawal [F(2,9) = 69.717; p < 0.001]. BDNF mRNA levels were significantly increased at both 20 hr
(+294 ± 7%) and 70 hr (+192 ± 29%) of opiate withdrawal
as compared with controls (p < 0.05) (Fig.
2B,C, Table 1). It should be noted that there was a
significant reduction in BDNF mRNA levels at 70 hr of withdrawal as
compared with 20 hr (p < 0.05) (Fig.
2B,C, Table 1), indicating a partial return toward
control levels.
In contrast to the increase in BDNF mRNA expression in the LC during
morphine withdrawal, densitometric analysis of film autoradiograms revealed no significant alterations in NT-3 mRNA levels at 2 or 6 hr of
withdrawal as compared with controls [F(2,8) = 1.029; p > 0.1] (Fig.
3A,C, Table 1). However,
significant reductions in NT-3 mRNA levels were found after the longer
opiate withdrawal periods [F(2,9) = 70.719;
p < 0.001] (Fig. 3B,C, Table 1).
Post hoc analyses indicated that there was a significant
decrease in NT-3 cRNA hybridization in the LC after 20 hr (49 ± 2.9%) and 70 hr (19 ± 1.3%) of morphine withdrawal
(p < 0.05) (Fig. 3C, Table 1). The
magnitude of this reduction was significantly smaller at 70 hr as
compared with 20 hr (p < 0.05) (Fig.
3C, Table 1).
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Figure 3.
Effect of precipitated opiate withdrawal on NT-3
mRNA expression in LC. A, Film autoradiograms of NT-3
mRNA expression in LC of control rats and of rats after 2 or 6 hr of
opiate withdrawal. B, Film autoradiograms demonstrating
the expression of NT-3 mRNA in LC of control rats and rats exposed to
20 or 70 hr of opiate withdrawal. C, Results from
analysis of film autoradiograms show that there are no alterations in
NT-3 mRNA levels at 2 and 6 hr of opiate withdrawal as compared with
control. In contrast, there was a decrease in NT-3 mRNA levels at 20 and 70 hr of opiate withdrawal as compared with control
(p < 0.05). Results are expressed as a
percentage of control values. Values are the mean ± SEM (SEM for
2 and 6 hr control group = ±4.4; SEM for 20 and 70 hr control
group = ±4.) *Significantly decreased from control.
**Significantly different from control and 70 hr. Scale bar, 500 µm.
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In film autoradiograms, trkB mRNA expression appeared to be
increased in the LC at both 2 and 6 hr of morphine withdrawal as
compared with controls (Fig.
4A). Densitometric
analysis demonstrated a significant increase in the hybridization
density for trkB mRNA after withdrawal
[F(2,8) = 6.71; p < 0.02].
Post hoc analyses revealed small but significant increases
in trkB mRNA levels at 2 hr (+11 ± 2.9%) and 6 hr
(+9 ± 2.3%) of opiate withdrawal (p < 0.05) (Fig. 4C, Table 1). In contrast, no changes in
trkB mRNA levels were observed at 20 and 70 hr of withdrawal
as compared with controls (p > 0.05) (Fig.
4B,C, Table 1).
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Figure 4.
Effect of precipitated opiate withdrawal on
trkB mRNA expression in LC. A, Film
autoradiograms demonstrating trkB mRNA expression in LC
of control rats and rats exposed to 2 or 6 hr of opiate withdrawal.
B, Film autoradiograms of trkB cRNA
hybridization in LC in control rats and rats exposed to 20 or 70 hr of
opiate withdrawal. C, Densitometric analysis of film
autoradiograms reveals an increase in trkB mRNA levels
at 2 and 6 hr of opiate withdrawal as compared with control
(p < 0.05), and no changes in
trkB mRNA expression at 20 and 70 hr of opiate
withdrawal as compared with control. Results are expressed as a
percentage of control values. Values are the mean ± SEM (SEM for
2 and 6 hr control group = ±1.1; SEM for 20 and 70 hr control
group = ±1.7.) *Significantly increased from control. Scale bar,
500 µm.
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As with trkB, there appeared to be increased trkC
mRNA levels in the LC at 2 and 6 hr of morphine withdrawal as compared
with controls (Fig. 5A).
Densitometric analysis of film autoradiograms confirmed that there were
significant changes in the hybridization density for trkC
mRNA during withdrawal from morphine [F(2,8) = 17.09; p < 0.01]. Post hoc analyses showed
that trkC mRNA levels were significantly elevated at 2 hr
(+24 ± 3.8%) and 6 hr (+23 ± 3.7%) of morphine withdrawal
as compared with controls (p < 0.05) (Fig.
5C, Table 1). In striking contrast to the effects of early
withdrawal, significant reductions in trkC mRNA levels were
observed in the LC after longer withdrawal periods
[F(2,9) = 11.25; p < 0.05]
(Fig. 5B,C, Table 1). Levels of trkC mRNA were
significantly decreased at 20 hr (22 ± 1%) and 70 hr
(12 ± 4.2%) hr of withdrawal as compared with controls
(p < 0.05) (Fig. 5C, Table 1).
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Figure 5.
Effect of precipitated opiate withdrawal on
trkC mRNA expression in LC. A, Film
autoradiograms showing the expression of trkC mRNA in LC
of control rats and rats exposed to 2 or 6 hr of opiate withdrawal.
B, Film autoradiograms of trkC mRNA
expression in LC in control rats and rats exposed to 20 or 70 hr of
opiate withdrawal. C, Densitometric analysis of film
autoradiograms demonstrates an increase in trkC mRNA
levels at 2 and 6 hr of opiate withdrawal as compared with control
(p < 0.05), followed by a decrease in
trkC mRNA levels at 20 and 70 hr of opiate withdrawal as
compared with control (p < 0.05). Results
are expressed as a percentage of control values. Values are the
mean ± SEM (SEM for 2 and 6 hr control group = ±2.7; SEM
for 20 and 70 hr control group = ±3.7.) *Significantly increased
from control. Significantly decreased from control. Scale
bar, 500 µm.
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Ventral tegmental area
Chronic morphine
Coronal sections throughout the VTA of control and
morphine-treated rats were processed for the in situ
hybridization localization of BDNF, NT-3, trkB, and
trkC mRNAs. No changes in neurotrophin or neurotrophin
receptor mRNA levels could be detected in the VTAs of opiate-treated
rats as compared with controls by visual inspection of film
autoradiograms (Fig. 6). Densitometric
analysis of film autoradiograms confirmed that there were no
alterations in the expression of BDNF, NT-3, trkB, or
trkC mRNAs in the VTA after chronic morphine treatment
(p > 0.05) (Fig.
7). Similarly, there did not appear to be
any regulation of the neurotrophins or their receptor mRNAs in the
nearby substantia nigra (Fig. 6).
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Figure 6.
Film autoradiograms of BDNF (A, B),
NT-3 (C, D), trkB(E,
F), and trkC (G, H)
cRNA hybridization in the ventral midbrain of control (A, C, E,
G) and morphine-treated (B, D, F, H)
rats. Note that no significant alterations in neurotrophin or
neurotrophin receptor mRNA levels were observed in VTA after chronic
opiate exposure. SNpc, Substantia nigra pars compacta.
Scale bar, 1710 µm.
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Figure 7.
Analysis of BDNF (A), NT-3
(B), trkB
(C), and trkC
(D) mRNA expression in VTA after chronic opiate
treatment. No significant alterations in neurotrophin (A,
B) or neurotrophin receptor (C, D) mRNA levels
were observed in VTA with chronic morphine treatment. Results are
expressed as a percentage of control values. Values are the mean ± SEM.
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Morphine withdrawal
At 2 and 6 hr of morphine withdrawal, there were no significant
differences in the VTA for BDNF, NT-3, or trkC mRNAs as
compared with controls (p > 0.05) (Fig.
8A,B,D). However, a
small but significant effect of morphine withdrawal on trkB
mRNA expression was detected in this brain region
[F(2,9) = 5.094; p < 0.05].
Post hoc analyses showed a significant increase in
trkB mRNA hybridization in the VTA at 6 hr (+23 ± 3.9%) of morphine withdrawal as compared with controls
(p < 0.05) (Fig. 8C). Withdrawal had
no apparent effect on neurotrophin or trk receptor mRNA
levels in the substantia nigra (data not shown).
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Figure 8.
Densitometric analysis of neurotrophin (A,
B) and neurotrophin receptor (C, D) mRNA
expression in VTA during precipitated opiate withdrawal. No significant
alterations in BDNF (A), NT-3
(B), or trkC
(D) mRNA levels were observed in VTA during
withdrawal from opiates. C, trkB mRNA
levels were significantly increased as compared with control at 6 hr of
morphine withdrawal (p < 0.05). Results are
expressed as a percentage of control values. Values are the mean ± SEM. *Significantly increased from control.
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DISCUSSION |
The results of the present study demonstrate that chronic opiate
treatment and withdrawal differentially regulate neurotrophin and their
receptor mRNAs in the LC. Chronic morphine treatment led to small, but
significant, increases in BDNF and NT-3 mRNA levels and no changes in
trkB or trkC mRNAs in this brain region. Precipitated opiate withdrawal resulted in a robust increase in BDNF
mRNA expression at all time points examined. In contrast to BDNF,
there was a decrease in NT-3 mRNA levels at the more prolonged time
points (20 and 70 hr) of withdrawal. Although both trkB and
trkC mRNAs were increased at 2 and 6 hr of withdrawal, trkB mRNA had returned to control levels by 20 hr, whereas
trkC mRNA levels were reduced to below control values at the
20 and 70 hr withdrawal time points.
It is interesting to note that the modulation of NT-3 mRNA expression
in the LC during withdrawal was not immediate. Although BDNF,
trkB, and trkC mRNA levels were increased at 2 hr
of morphine withdrawal, decreased NT-3 mRNA expression was not observed
until 20 hr of withdrawal. There are precedents for a delayed decrease in NT-3 mRNA levels in the brain. For example, experimental brain trauma resulted in a significant increase in BDNF mRNA in the hippocampal dentate gyrus by 1 hr after injury, whereas NT-3 mRNA levels were not significantly decreased from controls until the 6 hr
time point (Hicks et al., 1997).
The early increase in trkC mRNA expression followed by a
decrease to below control levels in the LC during opiate withdrawal is
difficult to interpret functionally because of the fact that the probe
used recognized mRNA transcripts for both the catalytic and
noncatalytic isoforms of the receptor (Dixon and McKinnon, 1994; Albers
et al., 1996). Therefore, it is unclear whether the changes in
trkC mRNA detected in this study are caused by alterations in the levels of the catalytic or noncatalytic transcripts, or both.
Chronic opiate exposure led to a small but significant increase in BDNF
mRNA in the LC, with a robust increase observed during opiate
withdrawal. It has been hypothesized that BDNF and other neurotrophins
may in some cases be responsible for maintenance of neuronal phenotype
(Korsching, 1993; Lindsay et al., 1994). Within this context, one
possibility is that BDNF acts in a paracrine or autocrine manner in the
LC, which is plausible because LC neurons express both BDNF and
trkB mRNAs (Seroogy, 1994; Zhang et al., 1998). If so, the
increase in BDNF expression could serve to counter the effects of
chronic morphine and morphine withdrawal on the biochemistry of these
neurons. Future studies with antagonists to BDNF will be needed to
examine the validity of this hypothesis. Besides a potential paracrine
role for BDNF in the LC, recent evidence suggests that LC neurons can
anterogradely transport BDNF to their projection regions (Fawcett et
al., 1998). Because the LC has widespread projections throughout the
CNS (Lindvall and Björklund, 1983), this anterogradely
transported BDNF may be a trophic factor for many
neurotrophin-responsive neuronal populations. Increased expression of
BDNF in the LC during morphine treatment and withdrawal could then
contribute to adaptations and homeostatic mechanisms that occur in
these other brain regions as well.
Neurotrophins and their receptors have been observed to exhibit
plasticity after several manipulations and seem to be especially sensitive to alterations in neuronal activity. The increased neuronal activity observed with seizures leads to increases in BDNF (Ernfors et
al., 1991; Isackson et al., 1991; Rocamora et al., 1992;
Lindefors et al., 1995; Nibuya et al., 1995; Suzuki et al., 1995) and
trkB (Merlio et al., 1993; Lindefors et al., 1995; Nibuya et
al., 1995) and decreases in NT-3 (Rocamora et al., 1992; Suzuki et al.,
1995) mRNA levels in the hippocampus (for review, see Gall, 1993). It has been suggested that BDNF and its high-affinity receptor
trkB may play a role in neuroprotection (Lindvall et al.,
1992; Merlio et al., 1993) or in the long-term biochemical and
morphological changes associated with seizure (Gall, 1993). The early
increase in BDNF, trkB, and trkC mRNAs and the
delayed decrease in NT-3 and trkC mRNAs in the LC after
precipitated opiate withdrawal may be in response to the increase in LC
neuronal activity that occurs during withdrawal (Valentino and Wehby,
1989; Rasmussen et al., 1990). LC firing rate is increased within 3 min
of precipitated opiate withdrawal, peaks by 15-30 min, and returns to
control levels by 3 d (Rasmussen et al., 1990). Of particular
interest are the findings that expression of certain neurotrophins and their receptors are still significantly altered at 70 hr of withdrawal, a time at which all detectable withdrawal behaviors have dissipated and
the neuronal activity of the LC has returned to normal levels (Rasmussen et al., 1990). The persisting adaptations in neurotrophin systems could be involved, therefore, in more protracted aspects of
opiate abstinence, phenomena that are well described clinically but
have been difficult to detect in laboratory animals (Koob and Nestler,
1997). The hypothesis that BDNF induction in LC neurons during opiate
withdrawal is secondary to the increased firing of these neurons is
supported by the previous finding that administration of reserpine,
which also increases the firing rate of LC neurons (Melia et al.,
1992), induces a transient upregulation of BDNF mRNA within LC neurons
(Seroogy, 1994).
In contrast to the alterations observed in LC after chronic morphine
treatment or withdrawal, there was little regulation of the
neurotrophins or their receptor mRNAs within the VTA. In fact, the only
change observed was a small but significant increase in trkB
mRNA in the VTA at 6 hr of opiate withdrawal. Neurotrophin expression
within the VTA has previously been determined to be malleable in adult
rodents. BDNF mRNA expression has been observed to be transiently
increased in the VTA after 6-hydroxydopamine-induced (Numan and
Seroogy, 1994) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced (Hung and Lee, 1996) lesions. This evidence suggests that the lack of
changes in the VTA in the present study are not caused by an inability
of the neurons to alter neurotrophin expression. It may not be
surprising that little regulation of the neurotrophins or their
receptor mRNAs was observed in the VTA during opiate withdrawal.
Opiates have been shown to acutely inhibit the inhibitory GABAergic
neurons in the VTA, thereby activating the dopaminergic neurons
(Johnson and North, 1992). Although the electrophysiological activity
of dopaminergic neurons during opiate withdrawal is not known, there is
indirect evidence that the opiate-induced inhibition of the GABAergic
interneurons is reversed during withdrawal, leading to a pronounced
inactivation of the VTA dopaminergic neurons (Bonci and Williams,
1997). Despite the lack of regulation of the neurotrophins and
receptors themselves, however, previous work has shown a clear modulation of postreceptor signaling proteins in the VTA after chronic
morphine treatment (Russell et al., 1994; Berhow et al., 1996).
The results of the present study provide further evidence for the
involvement of neurotrophin systems in opiate addiction, particularly
in the LC. Although the exact physiological role played by these
changes in the neurotrophins and their receptor mRNAs during the course
of chronic morphine treatment and withdrawal will require further
research, these findings highlight the complex array of adaptations
that contribute to the drug-addicted state.
| |
FOOTNOTES |
Received July 20, 1998; revised Sept. 29, 1998; accepted Oct. 1, 1998.
This study was supported by grants from the National Institute on Drug
Abuse, by the Abraham Ribicoff Research Facilities of the Connecticut
Mental Health Center, State of Connecticut, Department of Mental Health
and Addiction Services (E.J.N., D.S.R.), and by National Institutes of
Health Grant NS35164 and National Alliance for Research on
Schizophrenia and Depression (NARSAD) (K.B.S.).
Correspondence should be addressed to Dr. Eric J. Nestler, Laboratory
of Molecular Psychiatry, 34 Park Street, New Haven, CT 06508.
| |
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Abstract
Introduction
