Mu and kappa opioid agonists modulate ventral tegmental area input to the ventral pallidum

Mu and kappa opioid agonists modulate ventral tegmentalarea input to the ventral pallidum

Igor Mitrovic* and T. Celeste NapierDepartment of Pharmacology and Experimental Therapeutics, Loyola University Chicago Stritch School of Medicine, Building

102, 2160 South First Avenue, Maywood, IL 60153, USA

Keywords: dopamine, GABA, nucleus accumbens, rat

Abstract

The ventral pallidum (VP) is situated at the convergence of midbrain dopamine and accumbal opioid efferent projections. Using

in vivo electrophysiological procedures in chloral hydrate-anaesthetized rats, we examined whether discrete application of m- [D-Ala2,N-Me-Phe4,Gly-ol5 (DAMGO)] or k± (U50488) opioid receptor agonists could alter VP responses to electrical stimulation of

ventral tegmental area. Rate suppressions occurred frequently following ventral tegmental area stimulation. Consistent with an

involvement of dopamine in this effect, none of the 12 spontaneously active ventral pallidal neurons recorded in rats that hadmonoamines depleted by reserpine responded to electrical stimulation of ventral tegmental area. Moreover, in intact rats, the

dopamine antagonist ¯upenthixol attenuated evoked suppression in 100% of the neurons tested; however, the GABAA antagonist

bicuculline was able to slightly attenuate the response in 50% of the neurons tested. These observations concur with our

previous studies in indicating that ventral tegmental area stimulation releases dopamine (and sometimes GABA) onto ventralpallidal neurons. Both DAMGO and U50488 decreased the inhibitory effects of ventral tegmental area stimulation. These effects

on the endogenously released transmitter differed from those seen with exogenously applied dopamine, for DAMGO did not alter

the ef®cacy or potency of microiontophoretically applied dopamine. Taken together, these observations suggest that theinteraction between DAMGO and dopamine does not occur at a site that is immediately postsynaptic to the dopaminergic input

within the VP, but rather that opioid modulation involves mechanisms governing presynaptically released dopamine. These

modulatory processes would enable ventral pallidal opioids to gate the in¯uence of ventral tegmental area dopamine transmissionon limbic system outputs at the level of the VP.

Introduction

Functional and anatomical evidences (for reviews, see Mogenson &

Yang, 1991; Napier, 1993) point to the strategic position occupied by

the ventral pallidum (VP) in brain circuits involved in limbic±motor

integration. It is now well appreciated that the VP is a common target

for ventral tegmental area (VTA) dopamine (DA; Napier et al.,

1991a; Klitenick et al., 1992), and nucleus accumbens (NAc),

g-aminobutyric acid (GABA) and opioid efferents (ZaÂborszky et al.,

1985; Zahm et al., 1985), and that the VP provides a major in¯uence

on the functional output of both the basal ganglia and the limbic

system (Zahm, 1989; Mogenson & Yang, 1991).

The in¯uence of these transmitters within the VP has been the

focus of several contemporary reports. Binding assays (Contreras

et al., 1987; for review see Napier et al., 1991a) and immunostaining

studies (Boundy et al., 1993; Luedtke et al., 1999) con®rm the

expression of both D1- and D2-like DA receptors within the VP.

Electrophysiological evaluations demonstrated that VP neuronal

activity is regulated by both subtypes (Maslowski & Napier, 1991;

Napier & Maslowski-Cobuzzi, 1994). GABAA receptors are found

within the VP (Churchill et al., 1990; Zilles et al., 1991), and GABA

agonists inhibit VP cell ®ring (Lamour et al., 1986; Napier et al.,

1991b; Chrobak & Napier, 1993; Johnson & Napier, 1997a, b). In

addition, opioid receptors are expressed within the VP (Moskowitz &

Goodman, 1984; Lahti et al., 1989; Hiller et al., 1994; Olive et al.,

1997) and ligands selective for the m, k or d opioid receptor subtypes

(MOR, KOR, DOR, respectively) regulate neuronal activity within

the VP (Mitrovic & Napier, 1995, 1996, 1998). It is of considerable

interest therefore to ascertain the relationship(s) among these

transmitters in the regulation of VP neuronal ®ring.

Opioids are effective neuromodulators throughout the central and

peripheral nervous systems and their ability to alter the effects of DA

both pre- and postsynaptically is well documented (Heijna et al.,

1989; for review see McGinty, 1999). The arrangement of the VTA±

VP and VTA±NAc-VP circuitry may provide a means by which the

accumbal opioid projection to the VP can `®ne tune' the VTA

dopaminergic input to the VP. To test this possibility, the present

study evaluated the ability of MOR and KOR agonists to regulate VP

responding to activation of the VTA. This was accomplished in three

electrophysiological studies in anaesthetized rats in which extra-

cellular recordings were performed from single VP neurons during

electrical stimulation of the VTA, and drugs were applied directly

into the local milieu of the recorded VP cell. The ®rst two studies

substantiated previous reports showing an involvement of endogen-

ously released DA in VTA-evoked responding, and revealed that

while rapid responding to VTA activation is independent of the NAc,

Correspondence: Dr T. Celeste Napier, as above.E-mail: [email protected]

*Current address: Departments of Physiology, Anatomy and Stomatology,and W.M. Keck Center for Integrative Neuroscience, University of CaliforniaSan Francisco, San Francisco, CA 94143, USA.

Received 12 July 2001, revised 20 November 2001, accepted 23 November2001

European Journal of Neuroscience, Vol. 15, pp. 257±268, 2002 ã Federation of European Neuroscience Societies

GABAergic in¯uences may still be involved. For the third study, we

compared the ability the MOR agonist, D-Ala2,N-Me-Phe4,Gly-ol5-

enkephalin (DAMGO), to in¯uence the effects of endogenously

released transmitters with its ability to in¯uence the effects of

exogenous (microiontophoretically applied) DA. A general synopsis

of some of these ®ndings was included in a review chapter by Napier

& Mitrovic (1999).

Materials and methods

Animal surgery

Animals were handled in accordance with procedures recommended

in Guide for the Care and Use of Laboratory Animals published by

the Institute of Laboratory Animal Resources, Commission on Life

Sciences, and the National Research Council.

Male, Sprague±Dawley rats (Harlan, Indianapolis, IN, USA)

weighing 280±350 g were anaesthetized with chloral hydrate

(400 mg/kg, i.p.; Sigma Chemical, St Louis, MO, USA). A lateral

tail vein was cannulated for i.v. administration of the anaesthetic to

maintain surgical level of anaesthesia. The rats were mounted into a

stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA)

with the nose piece set at ±3.3 mm. The skull was exposed and holes

were drilled through the skull overlying the brain regions of interest.

Stereotaxic coordinates used for recording from the VP were 0.5 mm

posterior to bregma (P), 2.5 mm lateral to the midline (L) and

7.3±8.3 mm below dura (V). A stimulating electrode was placed at

5.7 mm P, 1.0 mm L and 7.8±8.0 mm V for electrical activation of

the VTA. For the experiments in which the NAc was pharmacolo-

gically inactivated, the needle tip of a 0.1-mL Hamilton syringe

(Reno, NV, USA) was placed 1.8 mm anterior to bregma, 1.6 mm L

and 7.3 mm V. Throughout the experiment, the rat's body tempera-

ture was maintained at 35±37 °C with a thermostatically controlled

heating pad (Fintronics, Orange, CT, USA).

Micropipettes and drugs

A multibarrel pipette-microelectrode assembly (as originally

described by Crossman et al., 1974), was used for the microionto-

phoretic application of ligands, and extracellular isolation and

monitoring of the individual action potentials. This electrode

assembly provides stable recordings of VP neuronal activity and

consistent responses to microiontophoretically applied ligands (e.g.

Napier et al., 1991b; Mitrovic & Napier, 1995). For its construction,

single and ®ve-barrel glass pipettes (glass tubing purchased from A-

M Systems, Everett, WA, USA) were heat-pulled (Narishige PE-2

vertical puller, Tokyo, Japan) and broken back to 2±3 mm and

9±11 mm outer diameter, respectively. The single pipette served as

the recording microelectrode, and was glued in parallel to the ®ve-

barrel iontophoretic pipette so that the microelectrode tip extended

10±20 mm beyond the iontophoretic pipette tip. The recording

microelectrode and the current-balancing barrel of the iontophoretic

pipette were ®lled with 2% pontamine sky blue dissolved in a 0.5-M

solution of sodium-acetate. The remaining four barrels of the

iontophoretic pipette contained DAMGO [D-Ala2,N-Me-Phe4,Gly-

ol5]-enkephalin (a highly selective MOR agonist; Bachem, Torrance,

CA, USA; 10 mM in H2O), dopamine hydrochloride (DA; Sigma;

200 mM in H2O) or GABA (Sigma; 100 mM in H2O). In some

experiments, the GABAA antagonist bicuculline (5 mM in H2O,

Sigma) was loaded into the fourth barrel and tested. In other

experiments, U50488, trans-(6)3,4-dichloro-N-methyl-N-[2-(1-

pyrrolidinyl)cyclohexyl]-benzene-acetamide methane sulphonate (a

highly selective KOR agonist; Research Biochemicals International,

Natick, MA, USA; 200 mM in H2O) was tested. The impedances of

the recording electrodes were between 4 and 7.5 MW, when measured

in physiological saline at 165 Hz with a microelectrode tester

(Winston Electronics, San Francisco, CA, USA). Similarly measured

impedances of the iontophoretic pipettes ranged from 20 to 85 MWand those of the current balancing barrel ranged from 9±22 MW.

In experiments that were designed to assess the contribution of

GABA and DA to VTA stimulation-evoked responses, the DA

antagonist ¯upenthixol (Research Biochemicals International; 1 mg/

mL H2O) was injected intravenously (doses 0.5±4 mg/kg). To

inactivate the NAc, procaine hydrochloride (Sigma; 40 mg as the

salt/2 mL saline/2 min) was administered directly in the nucleus using

a carrier-mounted microdrive (Starrett, Athol, MA, USA) to depress a

0.1-mL syringe plunger. This protocol effectively inactivates the NAc

for up to 45 min without anaesthetizing VP neurons (Napier, 1992a).

To acutely reduce DA, reserpine (Sigma; 5.0 mg/kg i.p.; in H2O with

a few drops of glacial acetic acid added to improve solubility; ®nal

pH = 5) was administered 6 h before, and a-methyl r-tyrosine

(Sigma; 250 mg/kg i.p.; in a 20% ethanol solution) was administered

1 h before evaluating electrophysiological responses of VP neurons.

Microiontophoresis

The micropipette assembly was lowered in the rat's brain with a

hydraulic microdrive (Trent-Wells, South Gate, CA, USA). Spon-

taneous VP neuronal activity was sampled, and monitored with a

storage oscilloscope (Kikusui Electronics, Japan) and an audio

analyser (Grass Instruments, Quincy, MA, USA). Individual action

potentials were ampli®ed, ®ltered (200 Hz and 2 kHz) and isolated

from the background with an ampli®er/voltage discriminator

(Fintronics). The output was transferred to an IBM-compatible PC,

which, using custom software, generated online histograms and

quanti®ed the data. Drugs were ejected with a cationic current

ranging from 1 to 112 nA, and retained with a 10 nA anionic current,

using a six-channel current generator and programmer (Fintronics).

At the beginning of each electrode penetration (while dorsal to VP),

ejection currents were applied to each of the drug-containing barrels

for 30 min to concentrate the drugs at the pipette tip. Subsequently, a

spontaneously ®ring neuron was electrically isolated and a stable

baseline ®ring frequency and pattern were obtained. For those cells

with consistent interspike intervals, a stable recording was achieved

when ®ring variation was less than 15% for at least 5 min. Because

some VP cells ®re in a periodic (i.e. cyclic) pattern, stable ®ring was

considered to have occurred with these neurons only if the mean

®ring for each cycle of at least three cycles (or 5 min of recording,

depending upon which was longer), did not vary more than 15%.

Changes in neuronal activity were attributed to an ionto-

phoretically applied drug if the ejection currents of no more than

40 nA altered the ®ring rate by at least 20% from the pretreatment

rates during at least two drug applications (each with a duration of no

more than 3 min). Drug-induced effects were evaluated by comparing

neuronal ®ring, as averaged across the 30-s drug application period

with ®ring rate averaged for 30 s immediately preceding the ejection

of the drug. This assessment was aided by a visual inspection of the

real-time-rate histograms to evaluate post-treatment recovery of the

neuronal activity and to make sure that the changes in neuronal

activity that could not be attributed to treatment effects (such as

periodic ®ring) did not obfuscate the data analysis. With those

neurons where periodic ®ring pattern occurred, care was taken to

administer all treatments during the same phase of the cycle.

In some experiments, the ability of subthreshold ejection currents

of DAMGO to modify the VP rate effects of exogenously applied DA

was evaluated. First, the DA ejection current±response relationship

258 I. Mitrovic and T.C. Napier

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 15, 257±268

was determined using 30-s incremental ejection current levels of DA

(usually 2, 4, 8, 16, 32, 48 and 64 nA). The same DA ejection current

range then was reassessed while DAMGO was coapplied at the

opioid's subthreshold ejection current level. This microiontophoresis

protocol has been demonstrated to consistently provide reliable

measures of the drug-induced effects in VP neuronal activity, as well

as the shifts in the ejection current±neuronal response relationship

induced by an opioid coapplication (e.g. Mitrovic & Napier, 1995,

1996).

Stimulation paradigms and protocols for evaluating opioidmodulation

For electrical stimulation of the VTA, stainless steel concentric

bipolar electrodes (NEX-100; 0.5 mm outer diameter, 0.2 mm inner

diameter, with 0.5 mm exposed tips separated by 0.5 mm; David

Kopf Instruments) were used to deliver current generated by a Grass

S88 stimulator coupled to a Grass stimulus isolation unit (SIU 5), and

a Grass constant current unit (CCU 1). Single, 0.1-ms, monophasic

pulses of various strengths were applied at 1 Hz to stimulate the

VTA.

A peristimulus histogram was generated for each sample of 128, 1-

Hz stimulation epochs, using a bin width of 2 ms. The mean number

of action potentials (or spikes) per bin occurring during 40 bins

(80 ms) immediately preceding the stimulation was considered as the

baseline and the average spikes per bin for this time-period was used

to determine the prestimulus mean. The 50 bins (100 ms) immedi-

ately following the stimulation were analysed for the occurrence of

the evoked responses. The onset and offset of an evoked response

component (inhibition or excitation) were characterized for the 128

epochs during which no drugs were applied (control). The onset of

the evoked response was de®ned as the ®rst of three consecutive

poststimulus bins that differed from the prestimulus mean by more

than 1.26 SD; offset was de®ned by the ®rst of three consecutive bins

that no longer met this criterion. As described by Chrobak & Napier

(1993), this approach sets P at < 0.001.

To investigate the capability of iontophoretically applied DAMGO

and U50488 to modulate neuronal responses elicited by electrical

stimulation of the VTA, the following experimental protocol was

used. (1) A spontaneously active VP neuron was isolated and a stable

baseline ®ring rate was obtained. (2) The VTA was electrically

stimulated. (3) Neurons responding to VTA stimulation were tested

for sensitivity to iontophoretically applied opioid agonists. (4) For

opioid-sensitive neurons, the ability of the opioid to modulate the

VTA-evoked response was ascertained. Parameters used for agonist

modulation were determined as follows. (i) To determine the opioid

ejection current level, the current was decremented from a level that

produced a robust neuronal response to one where neuronal activity

was altered less than 20% from baseline ®ring rate (termed

`subthreshold'). This process was used for incrementally evaluated

currents to allow the determination of the ejection current level that

was required to produce 50% of the maximal rate suppression

(termed Ecur50). When the maximal suppression (i.e. Emax) was not

obvious `on line', the Ecur50 employed in subsequent on line

assessments was based on our previous study using the same

methodological approach to generate complete DAMGO and U50488

ejection current±response curves (Mitrovic & Napier, 1995).

Subthreshold and Ecur50 were the ejection current levels used for

all evaluations of opioid in¯uences on VTA-evoked responses. (ii)

VP responses to a range of VTA stimulation current magnitudes

(usually from 0.4 to 1.0 mA) were evaluated, and the stimulating

current that produced approximately half of the maximally evoked

response was used for subsequent evaluations. When possible,

4±12 min after termination of the agonist ejection current, electrical

stimulation of VTA was repeated to test for the recovery of the VTA-

evoked response.

To isolate opioid modulation of VTA-evoked effects, it was

necessary to ensure that even small opioid-induced alterations in

spontaneous ®ring did not confound the analysis and interpretation of

the effects on VTA-evoked events. This confounding variable was

eliminated by using a previously established method for data analysis.

This method predicts the number of action potentials that would

occur in an evoked response component during control (no drugs

applied) if the control baseline were the same as the one that occurred

during treatment (Chrobak & Napier, 1993; Maslowski-Cobuzzi &

Napier, 1994; Turner et al., 2001). This `expected number' (eN) was

obtained by multiplying the number of spikes occurring within an

evoked component of the control sample (N, no drug applied) by the

ratio of the prestimulus baseline rate obtained during a drug treatment

(dR) and the prestimulus baseline rate obtained in the control (cR; no

drug applied), i.e. eN = N(dR/cR). Predictably, when calculated for

neurons with interstimulus baselines that were not altered by the

treatment, the eN did not differ from the actual number of spikes

occurring within an evoked component during the control (examples

of these are provided in the legends for Figs 1 and 3). Thus, for

statistical analysis, the eN was calculated for all VP neurons tested

and to determine if drug treatment signi®cantly altered evoked

responses, the eN for control periods were compared with the number

of spikes that actually occurred in the same evoked component during

drug treatment using a paired t-test. Drug effects on VTA-evoked

responses were also evaluated `categorically', with a 20% change of

the number of action potentials within the VTA-evoked component

(with the 128 stimulation epochs) during drug treatment vs. the

control (no drug) used to indicate a `signi®cant' treatment effect.

Histological and biochemical evaluations

At the end of each experiment, an anionic current was passed through

the recording microelectrode to deposit pontamine sky blue and mark

the recording site. The animal then was deeply anaesthetized with

chloral hydrate, and perfused with 0.9% sodium-chloride. The brain

was removed from the skull and stored in a 4% formalin±20% sucrose

solution for ®xation. With rats that were pretreated with reserpine, the

striatum was dissected out and quick-frozen on dry ice before the rest

of the brain was placed in the ®xative solution. To verify the lesion

extent, the concentration of DA and two other monoamines

(norepinephrine and serotonin) were determined in striatal tissues.

Compounds were separated with high performance liquid chroma-

tography and detected electrochemically (HPLC + EC) as previously

employed by Napier and colleagues (Napier & Potter, 1989;

Heidenreich et al., 1995).

Fixed brains were cut with a cryostat±microtome (Hacker

Instruments, Fair®eld, NJ, USA). Sections containing blue dye

deposits, and microelectrode and stimulating electrode tracks, were

mounted on glass slides and stained with neutral red. The location of

the blue dot, as agreed upon by two people, served as a reference

point for stereotaxic localization of the remaining recording sites.

Similarly, the location of lesion induced by the VTA stimulation

electrode and its tip was ascertained and agreed upon by at least two

people.

Statistics

For analysis of drug effects on the VP neuronal activity, pretreatment

®ring rate was standardized to 100%. Drug effects are reported as a

percentage change from the pretreatment rate. Two-tailed Student's

t-test, one-tailed paired t-test and c2-test were used for the data

Opioid modulation of dopamine 259


comparisons with a P < 0.05 required for signi®cance. All

parametric data are presented as mean 6 SEM.

Results

Study Ia: Ventral pallidal responding to activation of theventral tegmental area

Upon histological veri®cation of the placements for the VTA-

stimulating electrode tips and VP recording sites, the effects of VTA

stimulation on 86 VP neurons from 37 rats were evaluated. Analysis

of recording electrode placement revealed that the entire rostrocaudal

as well as mediolateral extent of the VP and rostral extreme of the

sublenticular substantia innominata was sampled (for examples of the

histology and reconstructed recording sites, see Mitrovic & Napier,

1995). The recorded neurons exhibited spontaneous activity with an

average ®ring frequency of 14.4 6 1.0 spikes/s. The action potentials

of recorded neurons had a duration of 1.75 6 0.1 ms and an

amplitude of 0.8 6 0.1 mV. The majority (71%) exhibited biphasic

wave forms with initial negative de¯ections. These are similar to our

prior extracellular evaluations of VP neuronal spiking (e.g. Mitrovic

& Napier, 1995, 1996, 1998).

Ninety-®ve percent (82/86) of the neurons tested responded to

VTA stimulation (stimulating currents ranged from 0.4 to 1.5 mA).

Five responding neurons were possibly activated antidromically as

they were able to follow high-frequency (200 Hz) stimulation and the

evoked response occurred with constant onset latency; these neurons

were not analysed further. The remaining responding neurons

(n = 77) did not exhibit these characteristics and thus were

considered to be orthodromically activated.

Both inhibitory and excitatory evoked responses were observed, 52

inhibitory components were obtained from the 77 responding neurons

(Fig. 1A) and 34 components were excitatory. Complex responses,

consisting of more than one component, were evoked from nine VP

neurons. For the inhibitory responses, the onset latency was

9.1 6 1.8 ms and the duration was 32.0 6 3.0 ms. Although the

distribution of the onset latencies was unimodal with 51 of the 52

responses occurring within 24 ms from the stimulus onset (the one

exception had a latency of 86 ms); the onset of one-half of all

inhibitory responses occurred by the 6±8 ms bin. The onset latency of

the 34 excitatory response components was 13.1 6 2.5 ms with a

21.0 6 3.0 ms duration. The excitatory onset showed a unimodal

distribution similar to that obtained with the inhibitory responses;

82% of the excitations occurred within ®rst 24 ms following the

stimulus onset with an additional six responses occurring between 30

and 54 ms. The response categories were not associated with the VP

locale of the recorded neuron nor did they differ among the various

VTA stimulation sites. The orthodromically evoked responses were

subsequently evaluated to determine the modulatory propensity of

opioids (Study Ib); however, for clarity of presentation, it is bene®cial

to ®rst report the ®ndings of a parallel experiment (Study II) targeted

at determining the transmitter responsible for VTA-evoked effects on

VP cell ®ring.

Study II: Evaluations of the transmitter(s) mediating the ventraltegmental area-evoked responses in the ventral pallidum

Candidate transmitters for mediating VP responding to VTA

stimulation are DA (Napier et al., 1991a; Maslowski-Cobuzzi &

FIG. 1. Peristimulus-time histograms of ventral tegmental area (VTA)stimulation-evoked inhibition of the ventral pallidum (VP). All fourhistograms are taken from the same VP cell thus illustrating the stability ofrepeated sampling from the same neuron. Each histogram shows theaccumulated number of action potential (spikes) in 2 ms bins for 128stimulation epochs. VTA stimulation (0.4 mA) was applied at time zero;represented by the bin counting the stimulation artefact. (A) Control (nodrug). The statistically determined onset and offset of the evoked inhibitoryresponse are indicated by the arrows, and these are provided in histograms(B±D) for reference. (B) This histogram was generated while bicucullinewas microiontophoretically applied (20 nA) onto the recorded VP neuron.The GABAA antagonist did not attenuate the VTA-evoked response in thisneuron (though it did in half of the six VP cells tested with bicuculline, seeResults text). (C) Subsequently, the nucleus accumbens (NAc) wasmicroinjected with 40 mg/2 mL/2 min of procaine and VTA stimulation wasrepeated 1 min thereafter. The evoked inhibition was not blocked. (D) Last,the rat was injected intravenously with the D1/D2 antagonist, ¯upenthixol(0.5 mg/kg). When VTA stimulation was repeated 2 min later, the evokedinhibition was no longer present; indicated by *. Note that the prestimulusbaseline (spontaneous ®ring) was not altered by any of the three drugtreatment protocols.



Napier, 1994) and GABA (Van Bockstaele & Pickel, 1995;

Steffensen et al., 1998; Rodriguez & Hernandez, 1999) with the

latter potentially including the accumbal GABAergic projections. For

example, behavioural reports demonstrate that DA receptor activation

in the NAc likely decreases the release of GABA in the VP (Pycock

& Horton, 1976; Jones & Mogenson, 1980; Mogenson & Nielson,

1983; Swerdlow & Koob, 1984; Swerdlow et al., 1990). Concurring

with this interpretation, electrophysiological evaluations have dem-

onstrated that some (less than half) of the encountered VP neurons

show a picrotoxin-sensitive rate increase following intra-NAc DA

infusions (Yang & Mogenson, 1989). However, speci®c activation of

accumbal D1-like DA receptors increases Fos-like immunoreactivity

in NAc neurons that project to VP (Robertson & Jian, 1995). This

allows for the possibility that Ð potentially depending upon which

DA receptor subtype is engaged (i.e. D1 vs. D2) Ð VP GABA

release also can be enhanced by accumbal DA. To explore more

directly the involvement of accumbal GABA in the present experi-

ments, we evaluated VTA-evoked VP responding after pharmaco-

logical inactivation of the NAc by intra-NAc injections of procaine,

and during microiontophoretic applications of the GABAA receptor

antagonist bicuculline onto the recorded VP neuron. In the ®rst series

of experiments, 11 VP neurons were tested in ®ve rats and nine

responded to VTA stimulation. These were representative of the

recorded cells used in Study I, for the spontaneous ®ring rate equalled

12.5 6 1.2 spikes/s, and the action potentials had a duration of

2.1 6 0.2 ms and an amplitude of 0.7 6 0.1 mV (two-tailed

Student's t-test, P = 0.229±0.673). Similar to Study I, the majority

of the recorded neurons exhibited initially negative, biphasic action

potentials with the remaining recordings being initially positive

triphasic action potentials (c32 = 3.45, P = 0.327). The distribution

of the VTA-evoked response categories was also similar to Study I;

seven neurons exhibited rate inhibitions, two neurons showed rate

enhancements and two did not respond to activation of the VTA (as in

Study I, stimulating currents ranged from 0.4 to 1.5 mA; c22 = 4.23,

P = 0.121). Of the seven that showed VTA-evoked inhibitions, six

recordings were held long enough to test further. Bicuculline was

evaluated to assess a possible contribution of GABA to the observed

inhibitory responses. At the bicuculline ejection currents that we have

previously demonstrated to be capable of antagonizing the effects of

iontophoretically applied GABA (Chrobak & Napier, 1993), the

GABAA antagonist did not alter the VTA-evoked inhibitions in three

of the six responses tested (Fig. 1B). But bicuculline attenuated the

evoked response by more than 20% in the remaining three neurons

(not shown). The effects of intra-NAc procaine injections on VTA-

evoked VP inhibitory responses also were tested for four neurons.

Similar to the report by Yang & Mogenson (1989), the disruption of

the VTA-NAc-VP axis had no effect on two neurons (Fig. 1C) but it

did attenuate the inhibitory response by more than 20% in the two

other neurons. Thus, a portion of the VTA-evoked inhibitions may

re¯ect a participation of accumbal and/or nonaccumbal (e.g. direct

VTA) GABAergic inputs; however, GABAergic transmission at the

level of the VP often is not required for VP responding to VTA

stimulation.

Two separate approaches were used to ascertain the involvement of

DA in the VTA-evoked responses. In one, the ability of the D1/D2

antagonist, ¯upenthixol (0.5 mg/kg i.v.) to antagonize the VTA-

evoked inhibitions was tested for four neurons (one cell recorded per

rat). Flupenthixol completely blocked VTA-evoked inhibitions in

three neurons (see Fig. 1D) and attenuated the evoked response by

more than 20% in the one remaining neuron tested. This ®nding

agrees with our previous reports on the ability of intravenous

administration and microiontophoretic application of DA antagonists

to attenuate VTA-induced VP responding (Maslowski-Cobuzzi &

Napier, 1994). In another experiment, the ability of VTA activation to

alter VP ®ring was tested in four rats acutely depleted of endogenous

DA by a reserpine + a-methyl r-tyrosine pretreatment. To ascertain

lesion extent, striatal monoamine concentration was assayed using

HPLC + EC methods in these rats, as well as in four unlesioned age-

and weight-matched controls also anaesthetized with chloral hydrate.

Expressed as pmol/mg wet tissue, DA concentration in controls was

70.92 6 7.76 and this was decreased to 0.46 6 0.11 with the

reserpine pretreatment (i.e. a 99% decrease from control levels).

Norepinephrine was 1.11 6 0.2 in controls and 0.07 6 0.07 in

reserpinized striata; serotonin was 2.68 6 0.34 and 0.16 6 0.06,

respectively. From the monoamine-depleted rats, a total of 12 VP

FIG. 2. Illustrations of the rate-suppressive effects of DAMGO and itsability to attenuate ventral tegmental (VTA)-evoked inhibitions in theventral pallidum (VP). (A) A real time-rate histogram demonstrating aDAMGO-induced decrease in spontaneous ®ring. Horizontal bars indicatethe onset and offset of ejection current and the numbers above the barsindicate the iontophoretic ejection current magnitude. Dashed lines illustratethe time of VTA stimulation that allowed the generation of the peristimulustime histogram (PSTH) shown in (C). (B,C) These PSTHs show thecumulative spikes per 2 ms bins immediately preceding and following VTAstimulation (1.0 mA; indicated by the vertical bar at time zero) for 128stimulation epochs. The onset and offset of the evoked inhibitory response,as statistically determined for the control condition, are indicated by thearrows. (B) Control. A PSTH generated while all drugs were retained in themicroiontophoretic pipette. This evaluation is of the same cell as that in(A), but at a time preceding the illustrated rate histogram. Ten actionpotentials occurred in the evoked inhibitory component. (C) Responsesobtained during iontophoresis of a subthreshold ejection current of DAMGO(5 nA). Forty-®ve spikes occurred in evoked inhibitory components duringiontophoresis of DAMGO and this number was compared with thecalculated expected number (eN, see Materials and methods) of 18 (seeFig. 4A left).



neurons was recorded during VTA stimulation. These neurons had a

spontaneous ®ring rate of 11.5 6 2.5 spikes/s, with action potentials

of 0.47 6 0.1 mV amplitude and 2.1 6 0.3 ms duration. Eight

recordings were of biphasic action potentials; the rest were triphasic.

Five of the 12 exhibited an initially positive de¯ection of the action

potential, and the other seven were in the negative direction. These

characteristics were not signi®cantly different from those obtained for

normal rats in Study I (using two-tailed t-test and c2 analysis,

P = 0.252±0.865). However, in stark contrast to Study I where 95%

of the cells tested responded to VTA stimulation, none of the VP

neurons recorded in reserpinized rats were sensitive to electrical

activation of the VTA. This lack of responding occurred despite the

relatively high currents tested (1.5 mA). Thus, while these experi-

ments did not exclude a role for norepinephrine and/or serotonin, it

demonstrated that DA is (either directly or indirectly) critically

involved in the evoked responses obtained in the VP upon VTA

stimulation.

Study Ib: The effects of DAMGO and U50488 on VTAstimulation-evoked responses

To determine if opioids can modulate VTA in¯uences on the VP, 41

VTA-sensitive neurons from Study Ia were subsequently tested with

the MOR agonist DAMGO, and 24 were tested with the KOR agonist

U50488. Forty-nine percent of the neurons responded to DAMGO

and 67% to U50488. The two drugs almost exclusively inhibited

neuronal activity (a rate enhancement occurred in only two neurons

that were tested with DAMGO). As we reported previously (Mitrovic

& Napier, 1995), the magnitude of the suppression was directly

proportional to the magnitude of the ejection current. Our prior work

also demonstrated that the microiontophoretically applied agonists

retained their selectivity for their preferred receptor subtype in that

DAMGO-induced inhibitions were blocked by MOR antagonist

CTOP and not by the KOR antagonist norbinaltorphimine, whereas

U50488 was antagonized by norbinaltorphimine and not by CTOP

(Mitrovic & Napier, 1995). In 13 neurons, both DAMGO and U50488

were tested. A decrease in ®ring was induced by only one opioid in

38% of these neurons and 23% showed rate suppression in response

to both opioids. These proportions are consistent with our prior report

(Mitrovic & Napier, 1995).

After establishing the effects of the opioids on spontaneous ®ring,

DAMGO and U50488 were applied during VTA stimulation (see

Figs 2 and 3). Subthreshold current for DAMGO (1±5 nA) attenuated

the response magnitude of VTA-evoked inhibitions (Figs 2 and 4A

left); with 13 of the 14 neurons tested showing a greater than 20%

increase in the number of spikes that occurred in the inhibitory

component and an overall signi®cant one-tailed paired-t analysis

(t13 = 3.69, P = 0.001). Subthreshold ejection currents of U50488

(1±5 nA) had a similar effect (Figs 3 and 4B left); spiking during the

VTA-evoked inhibitions was increased by more than 20% in eight of

11 cells tested with a signi®cant one-tailed paired-t evaluation

(t10 = 2.45, P = 0.017). Ecur50 U50488 (15±20 nA) enhanced evoked

spiking by more than > 20% in six of 10 tested neurons with a

signi®cant one-tailed paired-t (t9 = 2.09, P = 0.033; Figs 3(D) and

4A right). Interestingly, Ecur50 DAMGO (20±25 nA) was a less

FIG. 3. Illustrations of the rate-suppressive effects of U50488 and its abilityto attenuate ventral tegmental (VTA)-evoked inhibitions in the ventralpallidum (VP). (A) A real time-rate histogram demonstrating U50488-induced decreases in spontaneous ®ring. Horizontal bars indicate onset andoffset of ejection current and the numbers above the bars show theiontophoretic ejection current magnitude. Dashed lines illustrate the time ofVTA stimulation that allowed the generation of the peristimulus timehistograms (PSTH) shown in (C,D). (B±D) PSTHs (all taken from the samecell as in A) showing the cumulative spikes per 2 ms bins immediatelypreceding and following VTA stimulation (1.0 mA; indicated by the verticalbar at time zero) for 128 stimulation epochs. The onset and offset of theevoked inhibitory response statistically determined for the control conditionare indicated by the arrows. (B) Control. This PSTH was generated whileall drugs were retained in the microiontophoretic pipette and was taken at atime that preceded the rate histogram illustrated in (A). Thirteen actionpotentials occurred in the evoked inhibitory component. (C) Responsesobtained during iontophoresis of a subthreshold ejection current of U50488(2 nA); 44 spikes occurred in the evoked inhibitory component and this wascompared with the calculated expected number (eN, see Materials andmethods) of 18 (see Fig. 4B left). (D)Responses obtained duringiontophoresis of an Ecur50 for U50488 (18 nA). By de®nition, this ejectioncurrent level decreased prestimulus baseline (compare D with B) as well asin spontaneous ®ring (shown in A). Fifteen spikes occurred during theevoked inhibitory component and this was compared with the eN of nine(see Fig. 4B right).



effective modulator; while spiking in the VTA-evoked response was

increased > 20% in seven of 10 neurons, the magnitude of this

increased spiking often was relatively small and actually decreased in

three neurons (Fig. 4A right); thus, the overall one-tailed paired t-test

was not signi®cant (t9 = 1.00, P = 0.171). As we controlled for the

possible in¯uence that Ecur50 DAMGO-induced suppression of

spontaneous ®ring might have on evaluations of opioid effects on

VTA-evoked responding (see Materials and methods), these evalu-

ations suggest that mechanisms other than those that are employed to

attenuate VTA-evoked inhibitions may underlie the rate-suppressing

effects of MOR opioids.

To further assess the effects of opioids on the VTA-evoked

inhibitory response pro®le, we also analysed the response onset and

duration (a two-tailed Student's t-test was used for all of these

evaluations). At the subthreshold ejection current levels, DAMGO

did not alter the onset latency of the VTA stimulation-evoked

inhibitions (control onset, 7.3 6 1.4 ms; onset during DAMGO,

5.8 6 1.4 ms; t13 = 0.67, P = 0.519). However, subthreshold

DAMGO shortened the response offset (see Fig. 2B and C; control

offset, 33.3 6 3.6 ms; offset during DAMGO, 22.2 6 4.7 ms;

t13 = 2.39, P = 0.038). Thus the duration of the VTA stimulation-

evoked response was decreased by subthreshold DAMGO. Applied at

the Ecur50, DAMGO did not change the onset or offset of the evoked

responses (t9 = 0.13, P = 0.895 and t9 = 1.34, P = 0.212, respect-

ively). U50488 presented a different pro®le, for although the KOR

agonist generally attenuated the magnitude of the VTA-evoked

inhibition (see Fig. 4B), it did not alter temporal characteristics of the

evoked response. This was true for both the subthreshold ejection

current level (control onset, 5.4 6 1.5 ms; onset during U50488,

6.4 6 1.5 ms; t10 = 0.47, P = 0.644; control offset, 40.9 6 7.6 ms;

offset during U50488, 31.3 6 4.3 ms; t10 = 1.10, P = 0.288) and

Ecur50 (control onset, 6.2 6 1.2 ms; onset during U50488,

8.7 6 2.4 ms; t9 = 0.93, P = 0.365; control offset, 31.3 6 4.4 ms;

offset during U50488, 24.6 6 6.3 ms; t9 = 0.87, P = 0.396).

The effects of DAMGO and U50488 were also tested with nine

neurons with rates enhanced by VTA stimulation. Seven neurons

were tested with DAMGO and two with U50488. Neither opioid

agonist consistently in¯uenced the evoked excitations, as potentia-

tion, inhibition and no effect all were observed (data not shown).

Study III: Effects of DAMGO on the neuronal responseselicited by iontophoretically applied dopamine

Results from Studies I and II demonstrated that DA contributes to the

VTA stimulation-evoked VP responding and that opioid agonists can

alter this effect. To indicate whether the opioid modulation required

presynaptically released DA, these results were compared with those

collected in a third study where the ability of DAMGO to alter VP

responding to exogenously (iontophoretically) applied DA was

evaluated.

Fifty-three spontaneously active neurons located throughout the VP

in 28 rats were tested with DA and/or DAMGO. These neurons

exhibited ®ring activity of 14.5 6 1.0 spikes/s, with an action

potential duration of 1.9 6 0.2 ms and amplitude of

0.65 6 0.17 mV. Action potentials from 31 neurons were biphasic

(largely with initially negative waveforms), and 22 exhibited initially

positive triphasic spikes. The electrophysiological characteristics of

these neurons were not different from those observed for the neurons

tested in Study I (c2 and two-tailed t-tests; P = 0.232±0.882),

suggesting that similar populations were sampled. Thirty-two neurons

were evaluated for responding to DAMGO and, similar to Study I, 63%

were sensitive (c12 = 1.03, P = 0.310), with a rate suppression

occurring in all but two neurons. Fifty-three neurons were evaluated

for responding to DA, and 74% of these were sensitive. Both increases

(seen in 59% of the responding neurons) and decreases (41%) were

observed. Incrementally applied DA generated ejection current±

FIG. 4. Line graphs illustrating the ability of DAMGO (A) and U50488 (B)to attenuate ventral pallidal (VP) rate decreases evoked by stimulating theventral tegmental area (VTA). Each pair of connected circles represents oneneuron. ctrl, control; these circles indicate the expected number of spikesthat occurred in the control (no drug) condition. (Note: as detailed in theMaterials and methods, to control for possible confounds of drug-inducedchanges in baseline ®ring, the expected number of spikes, `eN', wasobtained by multiplying the number of spikes occurring within an evokedcomponent of the control sample (N; no drugs iontophoresed) by the ratioof the prestimulus baseline rate obtained during a drug iontophoresis (dR)and the prestimulus baseline rate obtained in the control (cR; no drugapplied), i.e. eN = N(dR/cR), see Materials and methods). Circles in thesubthreshold (subthr) and Ecur50 columns represent the actual number ofcounts that occurred in the VTA-evoked inhibitory component during drugapplication, with the evoked component onset and offset de®ned in thecontrol condition. *P < 0.05; one-tailed paired t-test (see Results text foranalysis).



response relationships that mirror classical dose±effect curves (see

Figs 5 and 6). Emax for the inhibitory responses was 46 6 12% of

baseline (spontaneous) ®ring rate (i.e. a 64% decrease) and Ecur50

equalled 25 6 8 nA. For the excitatory responses, Emax was

86 6 13% above baseline and Ecur50 was 23 6 4 nA. This response

pro®le is similar to our previous reports of DA-induced effects on VP

cell ®ring (Napier et al., 1991b; Johnson & Napier, 1997a).

To determine if a single cell could respond to both DA and

DAMGO, 32 neurons were tested with both ligands. Of those, 50%

were sensitive to both DA and DAMGO, 21% were sensitive to only

DA, and 9% to only DAMGO. To assess the modulatory effects of

DAMGO on DA-mediated responses, we employed a previously

utilized experimental paradigm (Mitrovic & Napier, 1995) where the

ability of subthreshold ejection currents of DAMGO to alter the

ejection current±response curve generated by incremental application

of DA was evaluated (see Figs 5A and 6A). As both the rate increase

FIG. 5. Illustration of dopamine-induced rate suppression of ventral pallidalcell ®ring, and the inability of DAMGO to alter these responses. (A) A realtime-rate histogram of a neuron which had ®ring suppressed by dopamine(DA) and by DAMGO (not shown). This histogram also illustrates theprotocol used to evaluate the ability of a subthreshold ejection current ofDAMGO to alter the DA-mediated response. Horizontal bars indicate onsetand offset of ejection current. Upward de¯ections indicate each incrementof DA ejection current (2, 4, 8, 16, 32, 64 and 112 nA). To conserve space,14 min of continuous recording was excised from the illustration (indicatedby parallel bars). (B) Ejection current±response relationship for the sixneurons with suppressed ®ring rate was by incremental DA application,with and without DAMGO coiontophoresis. Baseline equalled the averagespontaneous ®ring rate determined for 30 s immediately preceding eachtreatment and standardized to 100%.

FIG. 6. Illustration of dopamine-induced rate enhancement of ventralpallidal cell ®ring, and the inability of DAMGO to signi®cantly alter theseresponses. (A) A real time-rate histogram of a neuron with dopamine (DA)-increased ®ring. This histogram also illustrates the protocol used to evaluatethe capacity of a subthreshold ejection current of DAMGO to alter the DA-mediated response. Horizontal bars ¯anked with lines indicate onset andoffset of ejection current. Upward de¯ections indicate each increment ofDA ejection current (2, 4, 8, 16, 32 and 64 nA). Also illustrated is that ahigher ejection current of DAMGO produced rate suppression. To conservespace, 16 min of continuous recording was excised from the illustration(indicated by parallel bars). (B) Ejection current±response relationship forthe seven neurons with incremental DA application-increased ®ring rate,shown with and without DAMGO coiontophoresis. Baseline equalled theaverage spontaneous ®ring rate determined for 30 s immediately precedingeach treatment and standardized to 100%.



and rate decrease categories had a signi®cant number of DA-sensitive

VP neurons, both response categories were evaluated for opioid

modulation. However, coiontophoresing DAMGO with DA failed to

alter DA-mediated responses in either direction (see Figs 5 and 6). In

the presence of subthreshold DAMGO (1±5 nA), Emax for DA-

induced suppressions was of 60 6 7% below baseline ®ring and

Ecur50 was 29 6 8 nA (one-tailed paired t-tests for DA alone vs.

DA + DAMGO, Ecur50, t5 = 0.30, P = 0.489; Emax, t5 = 0.42,

P = 0.343). For DA-induced rate enhancements, DAMGO (1±5 nA)

coapplication yielded an Emax of 76 6 9% above baseline and an

Ecur50 of 27 6 7 nA. These also were not different from the

excitatory response pro®le obtained with DA alone (one-tailed paired

t-test, Emax, t6 = 0.63, P = 0.269; Ecur50, t6 = 0.50, P = 0.314).

These results indicate that neither the ef®cacy nor the potency of

exogenous DA to in¯uence VP cell ®ring is altered by activation of

local MOR. Thus, it appears that the modulatory function of VP

opioids on DA transmission involves mechanisms that are employed

upstream to DA interacting with its receptor (e.g. alterations in

presynaptic DA release).

Discussion

This study reveals that VP MOR and KOR can regulate VTA

in¯uences on the VP and that this function involves endogenously

released DA. As very little is presently known about the functional

relationship between the VTA-VP and VTA±NAc±VP systems,

several features of this interaction may prove to be of considerable

importance for understanding the integration of information within

the brain's limbic system.

The ®rst aspect of this study con®rmed and extended our previous

report (Maslowski-Cobuzzi & Napier, 1994) on the in¯uence of VTA

activation on VP function. A high percentage of VP neurons are

in¯uenced by electrical stimulation of the VTA (98% of the tested

neurons responded in the aforementioned study and 95% in the

present study). The great majority of the initial evoked responses had

an onset latency of < 24 ms, and this was true for both VTA-evoked

inhibitions and excitations. This pro®le is reminiscent of that reported

for electrophysiological investigations of the nigrostriatal pathway

(Feltz & MacKenzie, 1969; Zarzecki et al., 1977; Wilson et al.,

1982).

The excitatory responses that were initiated within a few ms after

VTA stimulation may re¯ect an involvement of excitatory amino acid

release. Cortical efferents colateralize within the VP as they course

caudally towards the midbrain (Sesack et al., 1989). It is possible that

the short-latency excitations evoked in the VP following VTA

stimulation involve antidromic activation of these corticopetal ®bres

(Delgado-Martinez & Vives, 1993) to release excitatory amino acids

within the VP. Supporting this scenario, Wilson et al. (1982)

demonstrated with intracellular recordings of striatal medium spiny

neurons in rats surviving 2±4 days after decortication (to allow

degeneration of the descending axons of cortical efferents) that the

large-amplitude, short-latency excitatory postsynaptic potentials

(onset ranged from 1.5 to 6.0 ms) typically seen upon electrical

activation of the substantia nigra pars compacta were no longer

apparent. However, even after thalamic transections were also

performed, small invariant (thus likely monosynaptic) excitatory

postsynaptic potentials (onset ranged 4.8±18.5 ms) could still be

evoked by nigral stimulation (Wilson et al., 1982), consistent with an

antidromic activation of excitatory cortical efferents to the nigra that

send collaterals to the striatum. There also is compelling evidence for

the colocalization of glutamate within dopaminergic neurons (Van

der Kooy et al., 1981; Sulzer & Rayport, 2000) which may have

contributed to the rapid-onset VP excitatory responses to VTA

stimulation seen in the present and prior (Maslowski-Cobuzzi &

Napier, 1994) studies.

GABA is a plausible transmitter candidate for the rapid inhibitions

seen with VTA stimulation. In a study of nigral-pallidal dopaminergic

projections, Yoshida et al. (1972) obtained short-latency inhibitory

postsynaptic potentials in neurons of the entopeduncular nucleus

(analogous to the internal segment of the primate globus pallidus).

These authors suggested that stimulation of the substantia nigra also

antidromically activated nigral GABAergic efferents that invade the

entopeduncular nucleus to monosynaptically inhibit these pallidal

neurons. It is plausible that a similar phenomenon may underlie some

of the VTA-evoked inhibitions of VP neurons seen in the present

study. Also, there is evidence that over 30% of the ascending

projections from VTA to the NAc are GABAergic (Van Bockstaele &

Pickel, 1995). If such projections send collaterals to the VP, their

activation would explain the few bicuculline-sensitive, rapid-onset

VTA-evoked inhibitions that we observed in the VP.

While the release of amino acids may have contributed to short-

latency, VTA-mediated responses in the VP, it is not likely that they

were responsible for the entire evoked response. Several aspects of

the present results and our prior studies support this conclusion. (1)

The VTA-evoked VP excitations demonstrated a more variable onset

with longer duration than that which occurs when known glutama-

tergic inputs are engaged [i.e. short-latency excitations evoked by

activating the basolateral amygdala (Maslowski-Cobuzzi & Napier,

1994) or the subthalamic nucleus (Turner et al., 2001) that are

attenuated by excitatory amino acid antagonists]. (2) The average

duration of the excitatory component was 21 ms and the inhibitory

component was 32 ms. These are longer than what would be

predicted if only ionotropic receptors were activated. (3) There is a

high degree of mimicry observed for the effects of microiontophore-

tic applications of DA and the evoked responses obtained with VTA

stimulation (Maslowski-Cobuzzi & Napier, 1994). (4) Both inhibitory

and excitatory VTA-evoked responses were attenuated by DA

antagonists when they were administered either systemically

(Maslowski-Cobuzzi & Napier, 1994, and present study) or via

microiontophoresis (Maslowski-Cobuzzi & Napier, 1994). (5) In rats

depleted of brain DA, VTA stimulation was not able to evoke

responses in the VP.

If indeed DA is a mediator of VTA-evoked responses that are

initiated by 24 ms, this seems in con¯ict with the time-frame required

for the cascade of biochemical events that is often associated with

signal transduction for metabotropic/G-protein-linked receptors such

as DA. The D1- and D2-classes of DA receptors are often linked to

increases (via Gas) and decreases (via Gai/o) in cAMP, respectively.

As reviewed by Missale et al. (1998), in vitro assessments of cAMP-

dependent conductances typically indicate a transduction delay of

100 ms or more. However, most of these in vitro studies were

conducted at room temperature, which can slow the kinetics of the

activated enzyme systems. More rapid activation would be antici-

pated in our in vivo preparations where body temperature was

maintained in a physiological range. There is also compelling

evidence that DA receptors can modulate ionic conductances via

membrane delimited pathways such as Gbg regulation of ion channel

activity (for reviews, see Missale et al. 1998; Nicola et al., 2000).

Signal transduction mediated by the membrane-delimited pathways

of G-protein-linked ion channels is a slower process than that

obtained by activation of ionotropic receptors, but it is considerably

more rapid than signal transduction mechanisms that employ more

indirect routes requiring, for example, enzyme activation/synthesis



and subsequent phosphorylation of the channel. Clearly, additional

assessments of stimulation-evoked responding are needed to ascertain

if direct G-protein activation of ion channels underlies the effects of

DA in the VP. When making such comparisons, however, it is

prudent to be mindful of the possible in¯uences that the different

conditions of in vitro and in vivo environments (e.g. temperature,

relative amount of active subunits, etc.) can have on evaluations of

synaptic transmission.

A major objective of the present study was to ascertain the ability

of activated MOR and KOR to modify VTA/DA in¯uences within the

VP. The results indicate that both receptor subtypes are involved, but

with differing roles. Subthreshold ejection currents for U50488

attenuated VTA-evoked inhibitions without altering the onset latency

or duration of the inhibitory component, whereas the ability of

DAMGO to attenuate this VTA-evoked response included a short-

ening of the component duration. Another feature that distinguished

the two agonists occurred at Ecur50; U50488 retained its modulatory

capacity, whereas DAMGO often did not alter VTA-evoked inhib-

itions beyond its suppressant effects on spontaneous ®ring. As lower

ejection currents expel less ligand than do higher currents

(Salmoiraghi & Stefanis, 1967; Hicks, 1984), the DAMGO-induced

effects likely re¯ected a concentration-dependent mechanism.

Response dependency on agonist concentration also was observed

by Olive & Maidment (1998) with their evaluations of MOR-

mediated release of enkephalin in the VP. Using in vivo micro-

dialysis, these scientists demonstrated that 1±10 nM of morphine

increased enkephalin concentrations, whereas 100 nM±100 mM had

little or no effect. The present results concur with these ®ndings. For

the pipette con®gurations, drug concentrations and nA current range

used to iontophoretically apply a subthreshold amount of DAMGO

are reported to deliver nM quantities of ligands; a mM range is more

likely with Ecur50 DAMGO (e.g. Salmoiraghi & Stefanis, 1967;

Bradshaw & Szabadi, 1974; Hosford et al., 1981; Hicks, 1984).

Pallidal MOR are located presynaptically on striatopallidal enkepha-

linergic projections as well as postsynaptically on most VP

GABAergic efferent neurons (Olive et al., 1997). Olive and

colleagues proposed that at low concentrations of MOR agonists,

the consequence of activating local presynaptic mechanisms is most

prevalent, whereas responding obtained with higher agonist concen-

trations largely re¯ects the in¯uence of polysynaptic feedback loops

that are regulated by pallidal GABAergic projections expressing

MOR. This latter in¯uence was proposed to be suf®ciently robust so

as to negate or mask the presynaptic effect (Olive & Maidment,

1998). This proposal helps explain the differential responding we

observed with subthreshold and Ecur50 DAMGO on VTA-evoked

responses that involved accumbal GABAergic projections to the VP.

Accordingly, the response attenuation that occurred with low

concentrations of DAMGO may re¯ect DAMGO's preferential

activation of MOR on GABAergic inputs from the NAc that are

engaged by VTA stimulation. With higher (i.e. Ecur50) concentrations

of DAMGO, a postsynaptic MOR may be activated which inhibits VP

GABAergic projections to the VTA. If so, the lack of an effect of

larger ejection currents for DAMGO on VTA/DA in¯uences in the

VP may re¯ect a disinhibition of the pallidal GABAergic inputs to

VTA dopaminergic neurons, which overrides the effects of activating

intra-VP presynaptic MOR.

The present study indicates that additional presynaptic MOR are

involved in VTA-evoked VP responses which are independent of

GABAergic inputs. These responses would be readily blocked if

MOR located on DA terminals in the VP were responsible for a

presynaptic regulation of DA release. However, this possibility is

incompatible with previous electrophysiological data suggesting that

VTA dopaminergic neurons do not express MOR (Gysling & Wang,

1983; Johnson & North, 1992). An alternative possibility is suggested

by evaluations of striatal regions. Here dopaminergic terminals

express NMDA receptors (Gracy & Pickel, 1996) and activation of

presynaptic NMDA receptors facilitates DA release (Ochi et al.,

1995; Cheramy et al., 1998; L'hirondel et al., 1999). As VP

dopaminergic inputs are collaterals from projections that terminate

within striatal regions (Klitenick et al., 1992), it is reasonable to

assume a similar ultrastructural relationship exists within the VP. We

have observed that morphine can inhibit glutamatergic transmission

in the VP (Johnson & Napier, 1997b), thus it is plausible that low

concentrations of DAMGO are suf®cient to activate MOR that are

upstream to the glutamatergic synapse. Activation of these MOR

could inhibit glutamate release and prevent a glutamatergic facilita-

tion of DA release. Concurring with the idea that DAMGO attenuated

VTA-evoked inhibitions by acting upstream to the recorded VP

neuron, is the observation that responses to exogenously applied DA

(which bypasses the need to endogenously release the transmitter)

were not altered by coiontophoresis of DAMGO. This apparent lack

of postsynaptic modulation of DA is unique among the VP

transmitters tested thus far, for both stimulation-evoked release and

microiontophoretic applications of glutamate, GABA and sub-

stance P are modulated by subthreshold ejection currents of MOR

agonists (Mitrovic & Napier, 1996; Johnson & Napier, 1997b;

Mitrovic & Napier, 1998).

In sum, the dissimilar features of DAMGO- and U50488-mediated

suppression of VTA stimulation-evoked responses suggest that KOR

and MOR differentially modulate the VTA transmission. This

explanation has support in ®ndings obtained from studies in the

NAc. Accumbal KOR are found almost exclusively on axon terminals

(Meshul & McGinty, 2000) and KOR agonists decrease accumbal DA

release (Heidbreder & Shippenberg, 1994). It is reasonable to

speculate that KOR on local DA terminals can regulate DA release

in the VP as well. This scenario is supported by the nature of the

suppression of the VTA-evoked responses by U50488 in the present

study. U50488 attenuated the evoked inhibitions without altering

onset or offset (i.e. duration) of the evoked responses, just as one

might predict if the underlying mechanism was an inhibition of local

neurotransmitter release; in contrast, DAMGO attenuated the

response duration. Moreover, DAMGO, but not U50488, appeared

to lose its modulatory function at higher (Ecur50) ejection current

levels, suggesting that the postsynaptic effects that render higher

DAMGO concentrations ineffective at modulating VTA-evoked

responding do not apply for the KOR receptor.

Behavioural reports provide insight into the functional conse-

quences of opioid modulation of DA neurotransmission in the VP.

Activation of VP dopamine receptors or MOR regulate several

aspects of motivated motor behaviours (Austin & Kalivas, 1990;

Hoffman et al., 1991; Klitenick et al., 1992; Napier, 1992b; Napier &

Chrobak, 1992; Hiroi & White, 1993; Johnson et al., 1993; Robledo

& Koob, 1993; Panagis & Spyraki, 1996; Gong et al., 1997, 1999; Xi

& Stein, 2000). In contrast, KOR do not appear to engage VP motor

systems (Hoffman et al., 1991) and effects of other VP-mediated

behaviours have not yet been assessed for this opioid receptor

subtype. The potential interaction of subthreshold doses for any of the

tested MOR or DA ligands also have not yet been evaluated

behaviourally. Nonetheless, direct intra-VP infusions of behaviou-

rally active (i.e. suprathreshold) doses of MOR or dopaminergic

agonists consistently are observed to increase locomotion in rats

(Austin & Kalivas, 1990; Hoffman et al., 1991; Klitenick et al., 1992;

Napier, 1992b; Napier & Chrobak, 1992; Gong et al., 1999). The

present study suggests this similarity in motor responding may occur,



at least in part, because VP concentrations of MOR agonists that are

high enough to alter ®ring no longer in¯uence DA mechanisms, thus

allowing the two transmitter systems to act in a similar `direction'

behaviourally. The present study also predicts that subthreshold

levels of MOR agonists (i.e. those too low to induce a measurable

behavioural effect) would antagonize the behavioural effects of DA

in the VP. As this theory illustrates, the ability of MOR to

`bimodally' regulate DA transmission to the VP may afford the

NAc (primary source of endogenous opioids to the VP) an important

means to ®ne-tune the behavioural output of the ventral striatopallidal

pathway.

Acknowledgements

The authors thank Drs Marta Margeta-Mitrovic and Toni Shippenberg for theirhelpful comments on this manuscript. U50488 was received as a gift from TheUpjohn Company (now Pharmacia). Work was supported by USPHSG#DA05255 to TCN.

Abbreviations

cR, control rate; DA, dopamine; DAMGO, [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin; DOR, d opioid receptor; dR, drug treatment rate; Ecur50, effectiveiontophoretic ejection current that produces 50% of maximal response; Emax,maximal effect; eN, expected number; GABA, g-aminobutyric acid;HPLC + EC, high pressure liquid chromatography with electrochemicaldetection; KOR, k opioid receptor; MOR, m opioid receptor; N, no drugapplied; NAc, nucleus accumbens; U50488, trans-(6)3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzene-acetamide methane sulphonate; VP,ventral pallidum; VTA, ventral tegmental area.

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Mu and kappa opioid agonists modulate ventral tegmental area input to the ventral pallidum

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