Dec 13, 1994 - at Univ of Illinois at Chicago Library on March 6, 2011 .... shafter et at. , 1987;. Wirtshafter and. Klitenick,. 1989). Inas- much as the. MR is one.
0022-3565/95/2731-0327$03.OO/O ‘FitE Jousru. OF PHARMACOLOGY um Expiw.tneru. Taapnrrics Copyright C 1995 by The American Society for Pharmacology .JPET
273:327-336,
and
Experimental
Vol. 273, No. Printed in USA.
Therapeutics
1
1995
Behavioral and Neurochemical Effects of Opiolds in the Paramedian Midbrain Tegmentum Including the Median Raphe Nucleus and Ventral Tegmental Area1 MARK
A. KLITENICK2
Department
and
of Psychology,
Accepted
for publication
DAVID
University
December
WIRTSHAFTER of Illinois
at Chicago,
Chicago,
Illinois
13, 1994
of morphine into the median raphe nucleus (MA) of rats produced a dose-dependent, naloxone sensitive increase in locomotor activity. Dose-dependent increases in activity also could be produced by intra-MR injections of the mu-opioid agonist Tyr-D-Ala-Giy-MePhe-Gly(oi)-enkephalin (DAMGO) and the delta-opioid agonist D-Pen2,D-Pen5-enkephalin (DPDPE), but not by the kappa-opioid agonist Dynorphin A (1-1 3). Mapping studies demonstrated that DPDPE produced larger responses when injected into the MR than into a number of adjacent structures, whereas the effective zone for obtaining responses with DAMGO appeared to extend forward into the caudal portion of the ventral tegmental area. The induction of hyperactivity by DPDPE and DAMGO was unaltered in animals with large depletions of forebrain serotonin produced by injections of 5,7-dihydroxytryptamine, suggesting that these effects
were
serotonergic mechanisms. Postthat serotonin turnover in the hippocampus was reduced slightly after intra-MR injections of DPDPE, but no effects were observed after injections of DAMGO or Dynorphin A (1-13). Injections of either DPDPE or DAMGO into the MA resulted in a large increase in dopamine turnover in the nucleus accumbens. Finally, intra-MA injections of DAMGO or Dynorphin A(1-13), but not DPDPE, stimulated ingestive behavior in nondepnved animals, although the effects were substantially smaller than those seen after injections of
Many studies have demonstrated that marked behavioral effects can be produced by manipulations of the MR. Electrolytic or excitotoxic lesions of the MR have been shown to produce dramatic increases in locomotor activity (Jacobs et at., 1974; Geyer et at., 1976; Lorens, 1978; Asin et at., 1979; Wirtshafter and Asin, 1982; Asin and Fibiger, 1983), ar’1 similar effects have been observed after intra-MR microinjections of the inhibitory GABAA agonist muscimol (Sainati and Lorens, 1982; Klitenick et at. , 1985; Wirtshafter et at.,
Wirtshafter,
Injections
Intra-MR
in-
jections of GABA agonists also result in large increases food and water intake by nondeprived animals (Klitenick
1987,
1988;
Wirtshafter
and
Trifunovic,
1992).
in and
enkephalin; performance
analysis
DYN,
Dynorphin
liquid
of variance;
A (1-13);
chromatography;
8-OH-DPAT,
DA, dopamine;
RVTA,
rostral
DOPAC, dihydroxyindolacetic 8-hydroxy-2-(di-n-propylamino)tetralin
through
indicated
muscimol. ioral and
These results neurochemical
demonstrate effects can
that pronounced behavbe produced by stimulation
of these is stimu-
lated.
the
1986,
MR
tivity
result and
rounding
1988,
1989).
in much
ingestive
and
larger
Lorens,
Injections effects
behavior
structures
(Sainati
than
such
as the
1982;
Paris
of muscimol
on
both
do
DR, and
injections
the
ac-
into
VTA
Lorens,
into
locomotor or the
1987;
surCMR
Klitenick
, 1988; Wirtshafter and Klitenick, 1989), sugthat these behavioral effects indeed result from an of muscimol in the MR, or its immediate vicinity, rather than through diffusion of the a ug to a distant site. Although intra-MR injections of muscimol have been shown and
Wirtshafte
gesting action
to reduce 1981; 1986),
V6T lZ3, Canada.
MA, median raphe nucleus; GABA, r-aminobutyric region lying caudal to the MA; 5-HT, 5-hydroxytryptamine (serotonin);
mediated
assays
of opioid receptors within the MA and that the pattern effects depends upon which opioid receptor subtype
Received for publication May 3, 1994. 1 This research was supported in part by National Institutes of Health Grant NS21359 (D. W.) and a National Research Service Award DA-05391 (M. A. K). 2 Present address: Department of Psychiatry, Division of Neurological Sciences, University ofBritish Columbia, 2255 Wesbrook Mall, Vancouver, B.C.,
ABBREViATiONS:
not
mortem
tive
hippocampal
Nishikawa neither behavior
pear nisms
5-HT
produced
to be mediated (Paris
Wirtshafter Although
turnover
and Scatton, the hyperactivity
and
(Forchetti
1985a,b; nor the
by intra-MR
entirely Lorens,
and Trifunovic, a substantial
muscimol
through 1987;
and
Wirtshafter increases
et at., in inges-
injections
serotonergic
Wirtshafter
1992). number of studies
Meek,
et
have
ap-
mechaat.,
1987;
now
exam-
acid; DA, dorsal raphe nucleus; VIA, ventral tegmental area;CMA, pontine DAMGO, Tyr-D-Ala-Gly-MePhe-Gly(ol)-enkephalin; DPDPE, D-Pen2,D-Pen5VIA;
CVTA,
caudal
VTA;
5,7-DHT,
acid; 5-HIM, 5-hydroxyindolacetic hydrobromide.
5,7-dihydroxytryptamine;
acid; HVA, homovanillic
HPLC,
high-
acid; ANOVA, 327
Downloaded from jpet.aspetjournals.org at Univ of Illinois at Chicago Library on March 6, 2011
ABSTRACT
328
and Wlrtshafter
Klltenick
Vol. 273
med the effects of manipulating GABA transmission within the MR, much less is known about the functional role of other transmitters within this structure. Several lines of evidence suggest that opioid substances may play an important role in the MR. For example, anatomical studies have demonstrated the presence within the MR of cells and fibers containing various opioid substances (Moss et at. , 1981; Robbins et at., 1982; Senba et at. , 1982; Smialowska et at. , 1985; Harlin et at. , 1987; Pollard et at. , 1989), and autoradiographic studies have indicated that the MR contains moderate to high densities 1977;
of several Moskowitz
types of opioid receptors and Goodman, 1985;
(Atweh
and
Kuhar,
studies
to a number of
locomotor
offorebrain activity
structures, both
in
intact
and
we conducted and
in
5-HT-
depleted
animals in order to investigate the role of serotonergic MR cells in mediating any observed effects. In order to directly investigate the effects of opioid receptor stimulation on serotonergic MR cells, we further examined the effects of intra-MR opioid injections on hippocampal 5-HT turnover. Dopamine metabolism in the nucleus accumbens and striaturn was also examined since previous studies have shown that intra-MR injections of several drugs can markedly alter forebrain DA turnover (Wirtshafter et at. , 1988, 1989).
Methods General Subjects.
Methods
were 173 adult, male, Sprague-Dawley-de300 ± 20 g obtained from a colony maintained by the University of illinois. Animals were housed individually in wiremesh cages and kept in a temperature and light controlled (12:12-hr rived
rats
Subjects
weighing
Blox,
cycle;
Chicago,
noted
lights
on 8:00 A.M.) environment.
below.
activity
and
were
conducted
ingestive
behavior)
and
at approximately
ad libitum (including
neurochemical
the
same
of the animals light:dark cycle. Cannula implantation. Surgery was
(Wayne
Food
IL) and water were available In all cases, behavioral testing
Lab-
except as locomotor
determinations time
the
during
light
phase
performed
with
were
standard
anesthetized
with Stainless-steel 22-gauge guide (Plastic Products, Roanoke, VA) were implanted stereotaxically so that the tips terminated 2 mm dorsal to either the RVTA (A/P, 2.2; D/V, -2.0; MIL, 0.0), CVTA (A/P, 1.0; D/V, 2.1; M/L, 0.0), DR (A/P, 0.2; D/V, -0.5; MIL, 0.0), MR (A/P, -0.2; D/V, -2.3; M/L, 0.0) or the midline CMR (AlP, - 1.4; DAT, -2.8; MJL, 0.0) according to the atlas ofPellegrino and Cushman (1967). The cannulas were first centered on the superior sagittal sinus and then, after retraction of the sinus (Wirtshafter et al., 1979), lowered into the brain. Acrylic stereotaxic
techniques,
sodium cannulas
while
(50 mg/kg
pentobarbital
cement
dental
animals
i.p.).
and stainless-steel the skull. The guide
cannulas
to
28-gauge
stainless-steel
to 1.5 mm
the
into
obturator
the brain.
NJ) was applied
screws
was
which
Sodium
topically
were
cannula
extended
sulfathiazol
to the wound
used kept
to anchor the patent with a
additional 1.0 & Co., Rahway, followed by a proan
(Merk
margins
of Flo-Cillin (0.25 mg s.c.; Bristol Laboratories, Syracuse, NY). Behavioral testing began after 8 days of postoperative recovery, during which time the subjects were handled daily. 5-HT depleting lesions. Intra-MR injections of 5,7-DHT creatinine sulfate, or its isotonic saline vehicle, were made in some of the rats prepared with cannulas aimed at the MR. Subjects were injected with desmethylimipramine hydrochloride (25 mg/kg i.p.) 45 mm phylactic
injection
injections which were made immediately ofthe cannulas. 5,7-DHT (7.5 p.g ofthe salt in 1.5 .d of saline) or its vehicle was injected into the MR over an 8-mm period after which the injection cannulas were left in place for an additional 3 miii to allow for diffusion. Behavioral experiments were
before
the
after
intracranial
implantation
begun
10 days
after
of postoperative
recovery.
Drugs. Aliquots of DAMGO, DPDPE and DYN (Sigma Chemical Co., St. Louis, MO) were keep frozen (-70#{176}C) until the day ofuse. All of the drugs used were prepared in normal saline except for DYN was
which VAT)
ci.
in a solution
dissolved
which
after
of methyl
the pH was raised et at., 1986).
, 1979; Spencer Intracranial
to 7.2
alcohol-0.1 with
NaOH
M HC1 (1/1, (Goldstein et
injections. Injections were made with a 28-gauge stainless-steel injection cannula connected to a Hamilton microsyringe by polyethylene tubing. The animal’s obturator was removed and the injection cannula was then inserted so that it extended into the
brain
for 2 mm beyond the end of the guide cannula. DAMGO, and DYN were administered in a volume of either 0.25, 0.5 or 1.0 .Ll at a rate of 0.25 pJ/min. The injection cannula was left in place for 30 sec following the injection after which it was removed, the obturator was replaced and the animal was returned to the test
DPDPE
environment.
were
Subjects
studies
involving
repeated
ferent
treatments
vened
between
tions)
which
hand
held
testing,
in a randomized successive
were
tests
conducted
day during the animals Locomotor activity
light tests.
during
individual
order (a
the
injections.
animals
and
maximum
at approximately
received
In all dif-
at least 3 days interof seven microinjecthe same time each
phase. Locomotor
activity
was
measured
in
of by a 9 x 9 array of holes 3.5 cm in diameter. Four infrared beams were positioned 3.5 cm above the floor in a 2 X 2 grid. Horizontal movements were registered every 4 mm on a counter located in an adjoining room. illumination was provided by two overhead 40 W fluorescent fixtures. The day before their first experiment, rats were given a sham microinjection and were proexposed to the photocell cages for 1 hour. Activity tests were coninfrared
which
photocell
were
ducted
habituation
and
71.5
measuring
x 71.5
X 27 cm, the
floors
perforated
by placing period
were
cages
returned
the
animals
in
which they to the photocell
after
the photocell cages received intracranial cages for a further
for a 1 hr injections 2 hr. The
Downloaded from jpet.aspetjournals.org at Univ of Illinois at Chicago Library on March 6, 2011
Herz, 1986; Mansour et at. , 1987, 1988). Several authors also have reported that lesions of the MR attenuate some of the effects of systemically administered opioids (Samanin et at. , 1970, 1973; Adler et at., 1975; Garau et at., 1975; Costall and Naylor, 1975; Glick and Cox, 1977; Chance et at. , 1978; Slater, 1981; Romandini et at. , 1986), and intra-MR injections of the opioid antagonist naloxone block morphine-induced analgesia (Romandini et at. , 1986). Additionally, systemic treatment with naloxone has been found to attenuate the catalepsy produced by acute intra-MR injections of kaiic acid (Przewlocka et at. , 1986). Finally, systemic treatment with opioid agonists has been found to alter 5-HT turnover in the hippocampus, a structure which receives much of its serotonergic input from the MR (Moore and Halaris, 1975; Steinbusch, 1981; Kohier and Steinbusch, 1982). In the current studies, we attempted to explore the functions of opioid receptors in the MR, in more detail than has been done previously, by examining the effects produced by intra-MR injections of specific opioid agonists. We utilized either morphine, the selective mu-opioid receptor agonist DAMGO (Handa et al. , 1981; Lutz et at. , 1985), the selective detta-opioid agonist DPDPE (Mosberg et at. , 1983) or the selective kappa-opioid agonist DYN (Wuster et at. , 1981; Chavkin et at. , 1982). The effects of these injections on locomotor activity and on food and water intake were examined, because previous studies have shown that these measures are highly sensitive to experimental manipulation of the MR (Coscina et at. , 1972; Asin and Fibiger, 1983; Klitenick et at., 1985; Klitenick and Wirtshafter, 1986, 1987, 1988; Wirtshafter et at. , 1987; Wirtshafter and Klitenick, 1989). Inasmuch as the MR is one of the major sources of serotonergic projections
Morris
light:dark
1995
Oploids
habituation
period
locomotor
(vide
activity
was
omitted
was
measured
in the for
neurochemical 44
min
studies
after
jection
where
a preweighed animals’ home
the
microinjections
infra).
catch measurements.
Histology.
Subjects
not
Rats
used
in
in which
biochemical
studies
were
per-
fused,
under deep followed by skulls and stored sections were then were mounted on stained with cresyl
sodium pentobarbital anesthesia, with normal 10% formalin. The brains were removed from the in formalin for at least 1 week. Frozen 64-sm cut through the cannula tracts. Alternate sections chrome-alum-coated slides, air dried and then violet. Data analysis. Data were analyzed either by Student’s t tests or by ANOVAS followed, where appropriate, by analyses ofcontrasts to identify the origin of significant overall effects. Data analysis was accomplished by using SYSTAT statistical software. saline
spillage
amount offood (Wayne cage, a sheet of paper
and
a graduated
drinking
329
Tegmentum
LabBlox) was placed in placed underneath it to
tube
provided.
Food
intake
was measured to the nearest 0. 1 g and water intake to the nearest 1.0 nil for a 2-hr period after injections, after which food and water were available ad libitum. Neurochemical effects of intra-MR opioid agomats. Three groups of 9 to 11 subjects were used to examine the biochemical effects produced by intra-MR injections of DYN (10 jig), DPDPE (10 jig) or DAMGO (437.5 ng) and three groups of nine vehicle-injected animals served as controls. Animals were removed from their home cages, given the appropriate injections and were then placed imme(corrected
diately
for
spillage)
in the activity
sacrificed
and
cages for a period
assayed
as
described
of44 mm. Subjects
were then
above.
Results Histology. in figure priate trated las
Representative 1. All
of the
structures previously
aimed
at locations (Wirtshafter
at the
at the
level
nuclei
of Gudden.
central
aspect
MR
of the
pontine Sylvius posterior
and
rostral CVTA
of
RVTA placements lar nucleus near CMR
cannula
cannulas
similar and DR
within
to those Klitemck,
terminated
portions placements
the
placements
terminated
we have
ventral
terminated
interpeduncular
shown appro-
1989).
within
of the
are the
nucleus,
illusCannu-
these
nuclei
tegmental dorsal
to the
whereas
were located rostral to the interpeduncuthe caudal pole of the mamillary bodies.
cannulas raphe
terminated within or slightly dorsal to the nuclei. Cannulas aimed at the aqueduct of terminated within the aqueduct at the same anterolevel
as
the
MR
and
DR
placements.
Protocols
Experimental
Locomotor 5 to 11 subjects tions 54.7,
effects of selective opioid agonists. Ten groups of were used to examine the locomotor effects of injecofDPDPE (0.625, 2.5, 5, 10 and 20 p.g) and DAMGO (13.7, 27.3, 109.4, 218.7 and 437.5 ng) into the RVTA, CVTA, DR, MR and
CMR.
Additionally,
a group
offour
subjects
with
cannulas
terminat-
ing in the cerebral aqueduct dorsal to the DR were tested after injections of DPDPE. Another group of six subjects with cannulas aimed at the MR was used to examine locomotor activity after injections of DYN (0.875, 2.5, 5, 10 and 20 jig). Locomotor effects of intra-MR morphine. Locomotor activity was
measured
morphine
in
five
at doses of0.1,
subjects
who
received
intra-MR
injections
0.5, 1.0, 5.0 or 10.0 pg in a volume
of0.5
of
p1.
Another group of four subjects received systemic injections of the opioid antagonist naloxone (1.0, 5.0 or 10.0 mg/kg i.p.) immediately before being given intracranial injections of morphine (5.0 jig). Effects of 5,7-DHT lesions on the locomotor response to opioid agonists. Ten subjects with 5,7-DHT lesions of the MR and eight vehicle-injected animals received tests of locomotor activity after intra-MR injections ofDYN (10 jig), DPDPE (10 jig) or DAMGO (437.5 ng). Testing began 10 days after production ofthe lesions. One week after the final test, animals were sacrificed as described above and levels of 5-HT and 5-HJAA were measured in the hippocampus to verify the effectiveness of the lesions. Ingestive behavior. Nine rats were used to study the effects of intra-MR injections of opioid agomsts on ingestive behavior. Food and water were removed from the animals’ home cages to avoid the possibility testing.
that
a subject
Animals
received
might injections,
have
just
taken
in a randomized
a meal
2 hr before order,
of vehi-
cle, DYN (10 jig), DPDPE (2.5 and 10 jig), DAMGO (54.7 and 437.5 ng) and the GABAA agornst muscimol (25 ng). During the microin-
Fig.
1. Camera lucida drawings of cannula placements in the AVIA (a), (b), MA (c), DA (d) and the CMA (e). All injection cannulas terminated within the darkened areas. Abbreviations: B, decussation of the brachium conjunctivum; C, inferior colliculus; d, DA; i, interpeduncular nucleus; L, medial lemniscus; m, MA; S. substantia nigra; V, sensory trigeminal nucleus.
CVTA
Downloaded from jpet.aspetjournals.org at Univ of Illinois at Chicago Library on March 6, 2011
biochemical assays were conducted were sacrificed by cervical fracture and their brains were removed rapidly and placed on an ice-cold glass block. The brainstems were separated from the rest ofthe brain and stored in formalin for at least 2 weeks, at which time they were prepared for routine histological examination of the cannulae placements. The hippocampi were removed by gross dissection and the remaining forebrain was frozen rapidly and mounted in a cryostat. One-millimeter thick coronal sections were taken through the striatum at the level of the anterior commissure and 0.75-mm thick sections were taken through the nucleus accumbens. Stainless-steel punches were then used to take bilateral samples ofstriatal and accumbens tissue, as we have described previously (Wirtshafter et at. , 1989). All tissue samples were homogenized in mobile phase described by Kilts et al. (1981) containing N-methyl-5-HT as an internal standard with the pH adjusted to 3.9. The homogenates were then centrifuged and aliquots of the supernatant were stored at -70#{176}Cuntil they were assayed by HPLC with electrochemical detection by using the method described by Kilts et al. (1981). The technique used allowed for the measurement of DA, DOPAC, HVA, 5-HT and 5-HLAA. The protein content of the remaining pellets was measured by the method of Lowry et at. (1951). Neurochemical
in the Midbrain
330
Klltenick
Locomotor after
and Wlrtshafter response
scores
DPDPE.
to
intracranial
Vol. 273
activity
Locomotor
microinjections
ofDPDPE
Cl)
C
(0.625-20
A
3200
-*-
RTA O.f TA
-.--
,.Lg) are
shown
injections
in figure
2 where
a larger
effect
had
it can on
be seen
locomotor
that
2500
intra-MR
did of the signif-
activity
(fig. 2A). Analysis ANOVA indicated icant effects ofcannula placement [F(5,42) = 25.2, P < .0001] and dose [F(5,210) = 13.84, P < .001] and a significant placement x dose interaction [F(25,210) = 9.2, P < .001]. Analysis of contrasts indicated that the four highest doses of DPDPE elicited significant increases in activity, compared to vehicle, after injections into the MR (P < .001). A small, but significant (P < .001) increase in activity also was seen after injections
data
into
the
by using
intra-CVTA
The
sites
injections
time
courses
are
of the
shown
drug
maximal
of the
highest
dose
locomotor
in figure effects
of DPDPE
responses
2B,
where
occurred
(20
1600 1200
F
be seen
the
during
first
500 400 0
DAMGO
175
C
125
C-)
that
8-min
100 75
U 50
0 0
response to DAMGO. Figure 3A shows total locomotor activity counts elicited in the 2-hr period after intracranial injections of DAMGO (13.7-437.5 ng). Adniinistration of DAMGO into either the MR or the CVTA appeared
25
-C
-#{149}0--*--
0.
.60
Cl)
C
-a-
RVTA
2400
-0-
C
40
60
50
100
ng
437.5 Vehicle 54.7 ng
120
CVTA
175 150
0 0
0
-C
_2000
0. .C
0 0
20
5) U
OR MR
U
0
4TH VENT
C
=.
.20
0
CVTA
-0-
-40
0
* 2500
MR
150
Locomotor
3200
(ng)
0
postinjection.
A
B
Cl)
jig).
to intra-MR
it can
MR
*
2000
100 125 75 -60
1600
-40
-20
0
20
TIME
40
60
50
100
120
(mm)
1200
Fig. 3. A, total activity scores after intracranial injections of DAMGO into the AVTA (n = 5), CVTA (n = 7), DA (n = 7), MA (n = 6) and CMR (n = 7). Vehicle is represented as 0. < .001 for both CVTA and MA vs. vehicle-treated animals at the same injection site. The time course of locomotor activity for the 1 -hr preceding and the 2 hr after injections of DAMGO into the MA and CVTA are shown in B and C, respectively.
-C
0.
0.625
0
2.5
10
DPDPE
B
20
For the sake of clarity, only the locomotor
(.tg)
of vehicle
MR
175
Cl) .
l C
125
=
100
-0--
vehicle
-‘---
5ug bug
-.--
150
5) U
0 0
75
.C 0.
50 25
.60
.40
-20
0
20
TIME
40
60
80
100
120
(mm)
Fig. 2. Effects of intracranial microinjections of DPDPE activity. Data are represented as mean ± S.E.M. A, total for the 2-hr period after injections Into the AVIA (n = 8), 4th ventricle (VENT; n = 4), DA (n = 6), MA (n = 9) and Vehicle is represented as 0. * < .001 vs. vehicle-treated
on locomotor activity counts CVTA (n = 1 1), CMA (n = 9). animals at the activity for the I -hr
same injection site. The time course of locomotor preceding and the 2-hr after injections of DPDPE are shown the sake of clarity, only the locomotor response and 1 0-jig dose of DPDPE are illustrated.
after vehicle
in B. For and the 5-
and two doses
of DAMGO
activity
after microinjections
(54.7 and 437.5 ng) are presented.
to elicit equivalent hyperactivity, whereas injections at the other sites were without effect. Analysis of these data by means of a repeated measures ANOVA revealed a significant effect of cannula placement [F(4,27) = 31.7, P < .0001] and dose of DAMGO [F(6,162) = 25.98, P < .0001] and a significant interaction between placement and dose [F(24,162) = 6.9, P < .001]. Post-hoc comparisons indicated that injections ofall but the lowest dose ofDAMGO (13.7 ng) into the MR or CVTA led to significant hyperactivity compared to vehicle treatments (P < .01). The locomotor responses produced by injections at these two sites did not differ significantly from each other at any of the doses tested (P > .2). In contrast, injections into the RVTA, DR or CMR did not produce significant increases in activity compared to vehicle injections (P> .05 for all comparisons). Figure 3, B and C shows locomotor activity across time in response to DAMGO injections into the
MR
(fig. 3, B and
3B)
and
CVTA
(fig.
3C).
It can
be
seen
from
C that the motor-stimulant response to injections at either site occurred with very short latency. As a measure of rise time, we calculated, for each rat with MR or CVTA cannulas, the latency to the end of a time bin in which the subject’s activity level first equaled or exceeded 50% of figure
Downloaded from jpet.aspetjournals.org at Univ of Illinois at Chicago Library on March 6, 2011
injections the
tested measures
other
a repeated
*
2400
than
1995
Oplolds
331
Tegmentum
A
* 2100
*
*
1800
(I)
In the Midbrain
U)
CU)
*
C 1500
00
011200
-C
-I.900
2” 0.
0
1’1 0 -C
600
0.
0.
300
0
0.1
0.5
1
awTnc.
.
LECN
10
(jig)
Treatment
B
B
C
2100
.2
8 .
5).... 1800
CO
U)
I 6
a) 4
1200 GJC
0 #{149}0 C
U oC41
-
0
,.
600
.
0.
5.HT 300
0
Fig. 4. Effects of intra-MA ior. All rats received each
5
1
Naloxone injections dose
10
(mg/kg)
of morphine
of morphine
on locomotor
in a randomized
behavorder
at
(minimum) intertrial intervals. A, cumulative photocell counts the first 120 mm after morphine injection. Data are shown as ± S.E.M., n = 9. * < vs. vehicle injections. B, antagonism of intra-MA morphine (5 jg)-induced locomotor activity by peripheral during mean
administration
5. Vehicle naloxone. =
highest
of naloxone.
is represented
Data are represented as mean ± S.E.M., n as 0. P < .001 vs. group not receiving
4-mm activity score recorded for that animal and Klitenick, 1989; Wirtshafter et at. , 1993). For the 437.5-ng dose of DAMGO, this analysis yielded rise times of 5.33 ± 0.84 miii for MR placements and 7.43 ± 1.04 mm for CVTA placements. A Student’s t test conducted on these data indicated that these two latencies did not differ significantly [t(11) = 1.40, P > .5]. Locomotor response to DYN. Although intra-MR injections of DYN (0.875-20 p.g) resulted in a small trend toward increased activity (data not shown), an ANOVA indicated that over the range of doses examined DYN did not significantly influence locomotor activity [F(5,25) = 1.84, P > .14]. Locomotor response to morphine and naloxone. Activity scores for the 2-hr period after intra-MR injections of (Wirtshafter
5.HIAA
Fig. 5. A, activity counts for the 44-mm period after opioid injections into the MA of vehicle (CONTROL n = 10)- or 5,7-DHT-treated (LESION, n = 8) rats. B, levels of hippocampal 5-HT and 5-HIAA for animals receiving vehicle (CONTROL) or 5,7-DHT (LESION) injections. Data are represented as mean ± S.E.M. P < .001 vs. vehicle-treated animals.
4-day
the
2
(0.1-10 jig) are shown in figure 4A where it can be seen that morphine elicited a significant, dose-dependent increase in locomotor activity [F(5,20) = 2.809, P < .05] with the maximal effect being produced by the 0.5-j.ig dose. Figure 4B shows that peripheral administration of naloxone produced a dose-dependent blockade of the hyperactivity produced by intra-MR morphine (5.0 jig) [F(3,9) = 7.281, P < .01]. Effects of 5,7-DHT lesions on the locomotor response to intra-MR opioids. As can be seen in figure 5A, 5,7-DHT treatments failed to alter locomotor activity after intra-MR injections of DYN (10 jig), DPDPE (10 p.g) and DAMGO (437.5 ng). A 2 x 3 (5,7-DHT x opioid agonist) ANOVA conducted on these data indicated a significant effect of opioid agonist [F(2,32) = 12.76, P < .001], but not 5,7-DHT (F < 1). The 5,7-DHT x opioid agonist interaction also failed to approach significance (F < 1). Analysis ofcontrasts indicated that activity levels were significantly higher after injections of DPDPE or DAMGO than after injections of DYN (P < .001). In addition, a marked depletion in hippocampal levels of both 5-HT (84.6%) [t(15) = 9.86, P < .002] and 5-HJAA (79.4%) [t(15) = 10.76, P < .001] was produced by the 5,7DHT treatments (fig. 5B). morphine
Downloaded from jpet.aspetjournals.org at Univ of Illinois at Chicago Library on March 6, 2011
Morphine
5
0
332
Klitenick
and Wirtshafter
Vol. 273
TABLE
1 and water Intake after intra-MR agonists Data are represented as mean ± S.E.M., n
Food
injections =
g
ml
±
DYN (10
5.8 10.2
(25 ng)
Muscimol *
P
P
.05;
.5). levels of 5-HT, 5-HIAA and
ratios for animals receiving intra-MR opiof the three peptide treatments signifi-
5-HT ratio
or 5-HIAA levels (P was reduced slightly,
>
.3 in all cases). but
The
significantly,
Cs
> 0
C 0
0
I20PftC
Fig.
7. Levels of DA, DOPAC,
after
P
DAMGO
the
corresponding
metabolite
.001 ; P
0.3 1.0 1.2
±
(437.5
DAMGO
in 7B)
the after
(fig.
7A),
the
increase
in
the
ratio after DPDPE [t(18) = 4.81, P < .001]. In the nucleus accumbens (fig. 7B), no statistically significant changes were observed after injections of DYN. In contrast, injections of DPDPE led to a significant decrease in DA concentration [t(18) = 3.14, P < .01] and to an increase in the concentrations of DOPAC [t(18) = 4.11, P < .001] and HVA [t(18) = 3.06, P < .01]. Additionally, DPDPE injections reDOPAC/DA
(437.5
ng)
Cs
> 0 C
0
0
suited [t(18)
An
=
in significant 7.95, P