Behavioral and Neurochemical Effects of Opiolds in ...

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


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


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


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


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