Interactions between the mu opioid agonist DAMGO and substance P in regulation of the ventral pallidum

SYNAPSE 23:142-151 (1996)

Interactions Between the mu Opioid Agonist DAMGO and Substance P in Regulation of the Ventral Pallidum

IGOR MITROVIC AND T. CELESTE NAPIER Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of

Medicine, Maywood, Illinois 60153

KEY WORDS

ABSTRACT Substance P (SP) increases, and the mu-specific opioid agonist DAMGO decreases neuronal firing within ventral pallidum (VP) of the basal forebrain. This study investigated a possibility that some VP neurons are oppositionally co-regulated by SP and DAMGO using microiontophoresis combined with the extracellular electrophysiological recordings from chloral hydrate-anesthetized rats. SP altered DAMGO's ejection current- response curve, decreasing Em, and slope, and increasing the EcuSO (ejection current level at which 50% of the maximal response was obtained). The modulation was observed even at low ejection current levels that, when applied alone, were not sufficient to alter neuronal activity (i.e., subthreshold). Also, DAMGO altered the Em, and slope of SPs ejection current-response curve. DAMGO induced these effects even a t subthreshold ejection current levels. The responses to each peptide were blocked by a receptor-specific antagonist. These findings demonstrate that SP and mu-activating opioids antagonize each other's effects on VP neuronal firing. Thus, they may interact as physiological antagonists in the regulation of VP-associated functions.

Opioids, Substance P, DAMGO, Ventral pallidum, Iontophoresis

o 1996 Wiley-Liss, Inc.

INTRODUCTION Opioid and tachykinin neuropeptides regulate a num-

ber of physiological functions throughout the central nervous system, often invoking opposite effects on these functions. For example, in various experimental para- digms testing cognition, performance is facilitated by substance P (SP) and impeded by opioids (for reviews, see Decker and McGaugh, 1991; McGaugh 1989). While SP mediates noxious stimuli, opioids are powerful anal- gesics (for review, see Fields et al., 1991). Opioids have rewarding effects (for review, see Koob, 1992), which are thought to motivate repeated chronic self-administration of exogenous opiates such as heroin (see Di Chi- ara and North, 1992). SP antagonists attenuate behavioral consequences of withdrawal from chronic opiate administration (Johnston and Chahl, 1991), indicating that SP may counteract some of the repercussions of long-term opiate use.

Enkephalin and dynorphin opioids and SP are co- distributed throughout the basal forebrain, including the ventral pallidum (VP) (Groenewegen and Russchen, 1984; Marksteiner et al., 1992). The nucleus accumbens is likely the primary source of opioid and SP inputs to the VP (Zaborszky et al., 1985; Napier et al., 1995, respectively). All three major classes of opioid receptors are found within the VP (i.e., mu, delta, and kappa; 0 1996 WILEY-LISS, INC.

Lahti e t al., 1989; Mansour e t al., 1994; Moskowitz and Goodman, 1984; Pilapil e t al., 1987), as is the tachykinin NK, (SP) receptor (e.g., Kiyama et al., 1993).

Recently, VP opioid and SP receptors were function- ally characterized at the level of the single VP neuron (Chrobak and Napier 1993; Mitrovic and Napier, 199513; Napier et al., 1995). Microiontophoretically applied mu-, delta-, and kappa-selective agonists predomi- nantly inhibit spontaneous VP neuronal activity with the mu agonist DAMGO altering firing in a higher portion of the neurons than kappa and delta agonists (Mi- trovic and Napier, 1995b). In contrast, SP and the peptidase-resistant tachykinin DiMeC7 (both NK, agonists) increase VP neuronal activity (Napier et al., 1995).

There is a large overlap between the VP-associated behaviors (e.g., reward, cognition, and locomotion) and those regulated by opioids and tachykinins. Hubner and Koob (1990) showed that the lesions within the VP attenuate heroin self-administration by rats suggesting that the VP is involved in mediation of the hedonic effects of opioids. Additionally, microinjection of the mu opioid agonist DAMGO within the VP alters the pattern of intracranial self-stimulation in a manner consistent

Received October 17, 1995; accepted in revised form November 30, 1995

SUBSTANCE P AND DAMGO INTERACTIONS 143

with a potentiation of the rewarding effects of self-stimulation (Johnson et al., 1993). Improved performance on cognitive tasks is observed after intra-VP infusions of SP in rats (Gerhardt et al., 1992; Kafetzopoulos et al., 1986). The effects of opioids on locomotor activity, following the intra-W injections, are particularly well characterized. Strong motor activation is observed with mu-selective agonists (low pmolar doses are effective; Austin and Kalivas, 1990; Hoffman et al., 1991; Napier, 1992) and, to a lesser extent, with delta agonists (low nmolar doses are required; Austin and Kalivas, 1990; Hoffman et al., 1991). On the contrary, kappa-selective agonists do not elicit changes in motor behavior (Hoff- man et al., 1991). The tachykinin receptor agonist Di- MeC7 also evokes a slight increase in motor activity after intra-VP microinjection, although relatively high doses are needed (3.0 nmolhemisphere; Napier et al., 1995).

These behavioral and electrophysiological observations emphasize the physiological relevance of the opioid and tachykinin systems within the VP. Evidence from other regions of the central nervous system suggest that the opioids and SP may co-regulate physiological functions associated with the VP. To address this possibility at the level of the single VP neuron, the present study utilized electrophysiological procedures for in vivo extracellular recording combined with microiontophoretic applications of SP and DAMGO.

MATERIALS AND lMETHODS Subjects and surgery

Extracellular single neuron electrophysiological recordings were performed in chloral-hydrate anesthetized (400 mgkg i.p., Sigma Chemical Company, St. Louis, MO) male Sprague-Dawley rats (Harlan Labora- tories, Inc., Indianapolis, IN) weighing from 280 to 350 g. The rats were fixed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) with the nose piece set 3.3 mm below the horizontal plane. After a midline scalp incision was made, a 3 mm burr hole was drilled through the skull a t 0.3 to 0.7 mm P to bregma and 2.1 to 2.5 mm L from the midline. For maintenance of anesthesia, a lateral tail vein was cannulated. Body temperature was maintained a t 37°C with a thermo- statically controlled heating pad (Fintronics, Inc., Orange, CT).

Electrode-micropipette preparation A multibarrel pipette-microelectrode assembly (glass

tubing purchased from A-M Systems, Inc., Everett, WA) was used for the microiontophoretic ligand application and extracellular isolation and monitoring of the individual action potentials. Single and five-barrel glass pipettes were heat-pulled (vertical puller, Narishige PE-2, Tokyo, Japan) and broken back to desired tip size (2-3 Fm and 9-11 Fm, respectively). The single pipette served as the recording microelectrode, and was glued

in parallel to the five-barrel iontophoretic pipette so that the microelectrode extended 10-20 pm beyond the iontophoretic pipette. The current-balancing barrel of the iontophoretic pipette and recording microelectrode were filled with 2% pontamine sky blue dissolved in a 0.5 M solution of sodium-acetate. Of the remaining four barrels of the iontophoretic pipette, each contained one of the following: DAMGO [D-Ala2,N-Me-Phe4,Gly-o15]- Enkephalin (Bachem, Torrance, CA), a highly selective mu opioid agonist (see Corbett et al., 1993); substance P (Bachem), a NKI receptor-preferring tachykinin (see Regoli et a1 . , 1994); (2 s , 3 S) - cis - 2 - (diphenylmethyl) -N - ( (2 -met hoxyphenyl ) methyl)- 1 -azabicyclo [ 2.2.21 octan- 3-amine dihydrochloride (CP96345 Pfizer, Inc., Groton, CT) a selective NK, tachykinin receptor antagonist (Snider e t al., 1991; for review, see Regoli et al., 1994), and its inactive enantiomer CP96344 (Pfizer, Inc.) [(2R,3R)-&-2-(diphenylmethyl)-N-( (2- methoxyphenyl)-methyl)-l-azabicyclo[2.2.2loctan-3- amine dihydrochloridel (Snider e t al., 1991; for review, see Regoli et al., 1994). All ligands were dissolved in sterile deionized water. The electrode impedance, measured in saline at 165 Hz (microelectrode tester, Win- ston Electronics, San Francisco, CA), ranged from 3-7 megohms for the recording microelectrode; 8-18 megohms for the current-balancing pipette, and 30-85 megohms for ligand-containing pipettes.

The pipette assembly was lowered in the brain with a hydraulic microdrive (Trent-Wells, South Gate, CAI. Spontaneous VP neuronal activity was sampled 6.9-8.3 mm ventral from the brain surface, and monitored with a storage oscilloscope (Tektronix Inc., Beaverton, OR) and an audio monitor (Grass Instruments Co., Quincy, MA). Individual action potentials were amplified, fil- tered (200 Hz and 2 KHz), and isolated from the background with a n amplifierholtage discriminator (Fintronics). The output was transferred to an IBM- compatible PC, which, using custom software, generated on-line histograms and quantified the data. DAMGO and SP were expelled from the microiontophoretic barrels with various cationic currents and retained with a 10 nA anionic current using a current generator (Fintronics). At the beginning of each electrode penetra- tion (while dorsal to VJ?) ejection currents were applied to each of the ligand-containing barrels for 30 min to concentrate the ligands at the pipette tip.

Microiontophoretic protocols Microiontophoretic ligand application has been SUC-

cessfully used to study the effects of locally applied opioid and tachykinin peptides on the VP neuronal activity (Mitrovic and Napier, 199513; Napier e t al., 1995). Microiontophoresis administers ligands directly into the local environment of the recorded neuron. Thus, this technique provides for a rapid assessment of very discrete treatments for individually administered ligands as well as their potential interaction when

144 I. MITROVIC AND T.C. NAPIER

co-applied. To evaluate opioid- and tachykinin-induced effects on VP cell firing in the present study, four microiontophoresis treatment protocols were used. l) To determine if activity changes could be induced by a peptide, a ligand sensitivity protocol was used. For this protocol, DAMGO or SP was applied using an ejection current level of at least 30 nA, for duration of minimum 2 min, and this procedure was repeated a t least twice. A peptide-induced change was considered to have occurred if the firing rate was uniformly altered by at least 20% from the pretreatment rates for at least two repetitions. This microiontophoretic protocol has been determined to consistently provide reliable peptide-induced effects on VP neuronal activity (e.g., Mitrovic and Napier, 1995b). Neurons whose firing rate was altered in aforementioned manner are further in text referred to as “sensitive” neurons, and those whose firing during ligand application did not meet these criteria were cate- gorized as “non-sensitive.” 2) Some neurons determined to be sensitive to SP were examined further to evaluate the receptor involved in the SP-induced effect. This was accomplished with an antagonism protocol previously used to verify that DAMGO-induced effects in the VP reflect an activation of m u opioid receptors. For this protocol, a NK, antagonist CP96345 and its NK1-inactive enantiomer CP96344 each were applied alone for at least 2 min after which they each were co-iontophoresed with SP. Ejection current levels used to apply SP were those used for that neuron to determine its sensitivity to the tachykinin. 3) Dependency of the magnitude of the peptide-induced response on the magnitude of the ejection current was determined using an incremental application protocol. This approach continuously ejects DAMGO and SP in increments of 5 nA every 30 sec so to allow a rapid assessment of threshold, E,,,, EcuSO, and slope for the peptide-induced effects on VP neuronal activity. This protocol has been used previously to obtain these pharmacological parameters for VP responses to opioid (Mitrovic and Napier, 199513) and NKl (Napier et al., 1995) agonists. It was determined that a particular ejection current level produces the same effects whether it was applied as single ejection current level (as used in the sensitivity protocol) or a part of the incremental protocol (Mitrovic and Napier, 1995b). Further, prolonged applications (up to 8 min) of the peptide using a single ejection current level produce neuronal response plateaus (indicative of a “steady state”) that are in stark contrast to graded responses obtained with incremental ejection currents. 4) Because potential shifts in Em,, EcuSO, and slope may provide valuable information regarding mechanisms of potential interaction, incremental application of the two peptides a n interaction protocol was used to evaluate possible interactions between DAMGO and SP. The interaction protocol tested the ability of a single ejection current level of one peptide (termed the modulator) to alter ejection current-response curve of the other

(termed the principal agonist). The modulator was applied alone (for a t least 2 min) to obtain a steady state condition before initiating the incremental application of the principal agonist. Because DAMGO and SP elicit opposite effects on VP firing rate, it was possible that simple subtraction of these effects might be interpreted as an interaction between the two peptides. To control for this potential confound, the ejection current levels used to apply the modulator were those that produced only minimal change (i.e., a t or just above a 20% change from baseline) or no change in the neuronal activity (termed near-threshold and sub-threshold, respectively), as determined by the incremental application protocol. The exceptions being the neurons that were sensitive to only one peptide. These also were tested with the interaction protocol with the peptide that did not elicit a response being applied as the modulator using EcuSO and Em, ejection currents as determined in previous studies with DAMGO (Mitrovic and Napier, 1995b) and SP (Napier e t al., 1995). The ejection current of the modulator was terminated only after conclusion of the incremental application of the principal agonist and a return of the neuronal activity to the pre-principal agonist firing rate was obtained. The ejection current- response curve obtained for the principal agonist with the interaction protocol was compared to the one generated €or the principal agonist alone.

Histology 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 anesthetized with chloral hydrate, and perfused with 0.9% sodium-chloride. The brain was removed from the skull and stored in 4% formalin-20% sucrose solution for fixation. Fixed brains were cut in cryostat-microtome (Hacker Instruments Inc., Fairfield, NJ). Sections containing blue dye depos- its and microelectrode tracks were mounted on speci- men glass slides and stained with neutral red. After agreement by two persons on the locations of the blue dots, these placements were marked in stereotaxic maps (Paxinos and Watson, 1986). Using the blue dot site as a reference point and stereotaxic coordinates of the recorded neurons, their locations were recon- structed on the maps.

To describe the topography of the VP neurons with regard to their sensitivity to DAMGO, SP, or both, the VP was divided into five subregions as follows [the coordinates closely correspond to those of Paxinos and Wat- son (1986)l: 1) rostral VP immediately in front of the anterior commissure crossing at approximately 0.05 mm anterior to bregma; 2) the subcommissural VP at the level of the crossing of anterior commissure, 0.26 mm caudal to bregma; 31 the subcommissural VF’ at 0.40 mm caudal to bregma; 4) the rostral substantia innominata and the caudal VP at 0.80 mm caudal to


TABLE I . Responses of 63 VP neurons to microiontophoretically applied DAMGO and SP

Non-sensitive Agonist Sensitive'

DAMGO 29/63 (46%) 34/63 (54%) SP 31/63 (49%) 32/63 (51%) Both 20/63 (32%) 23/63 (37%)

'Sensitive is defined as a minimal 20% change in neuronal firing by 30 nA of iontophoretic current.

bregma; 5) the rostral substantia innominata and the VP at 0.92 mm caudal to bregma. The rostral three subregions were divided further in ventromedial and dorsolateral territories according to differences in the histochemical markers and circuitry profile (Groene- wegen and Russchen, 1984; Zahm and Heimer, 1990).

Data analysis and statistics For analysis of treatment effects on VP neuronal ac-

tivity, pretreatment firing rate was standardized to 0%. Ligand effects are reported as percentage change of the pretreatment rate. In the sensitivity protocol, ligand- induced effects were measured by comparing neuronal firing for the 15 sec period at the end of a ligand treatment with the 15 sec period that immediately preceded ejection of the ligand. In the antagonism protocol, SP effects were measured by comparing neuronal firing for the 15 sec period at the end of the SP application with the 15 sec period during the ejection of the CP96345 or CP96344 that immediately preceded application of the peptide. Neuronal responses during incremental application of DAMGO or SP were measured by comparing 15 sec at the end of each current increment with the 15 sec period that immediately preceded incremental application of the peptide. These types of analyses have been previously demonstrated to provide reliable indi- cators of the microiontophoresed ligand-induced changes in the VP neuronal activity (Mitrovic and Na- pier, 199513; Napier et al., 1995). For the interaction protocol, firing rates obtained during application of the principal agonist were compared to the rate 15 sec immediately preceding the incremental application (which is during the application of the modulator at a single ejection current level). This comparison allows for study of the interaction between the two peptides independent of a possibility that the modulator changed spontaneous neuronal activity.

The cubic regression method as described by Pitts et al. (1990), was used to determine Em, (maximal effect of an agonist) and EeuSO (ejection-current that elicited 50% of the maximal effect). Slopes of the current response curves were calculated using linear regression for the steep component of the individual current-response curves. The changes in Em, for one peptide that were induced by co-application of the other peptide, demonstrated a bimodal distribution clustering into two groups of the responses: 1) those exhibiting a 0-5% change, and 2) those with >lo% change. Thus, a peptide

interaction was considered to have occurred in the neurons whose Em,, was altered > 10%. Pearson correlation coefficients were calculated to determine the goodness- of-fit for regression of SP's and DAMGO's populational ejection current-response curves. Student's t-test, and paired t-test were used for the data compari- sons with a P 5 0.05 required for significance. All para- metric data are presented as mean 'r S.E.M.

RESULTS Extracellular characteristics of recorded

VP neurons Sixty-three spontaneously active VP neurons with a

firing rate of 12 ? 1 Hz were evaluated for sensitivity to both SP and DAMGO. The amplitude of the recorded action potentials was 420 i 36 pV, and the duration was 1.6 -+ 0.1 ms. Seventy-six percent of the VP neurons demonstrated biphasic action potentials, 22% were triphasic, and 2% monophasic. Initially positive- and negative-deflections of the action potentials were encountered in almost equal proportions. These characteristics did not distinguish among neurons sensitive to DAMGO, SP, or neurons non-sensitive to both treatments.

Neuronal responses to DAMGO and SP Twenty-nine VP neurons demonstrated firing rate

changes in response to microiontophoretically applied DAMGO and 31 responded to SP (Table I). DAMGO exclusively inhibited firing while SP elicited only excitations. These results are in accord with their respective dispositions as inhibitory and excitatory neuropeptides (for review, see North, 1993; and Nakajima et al., 1991, respectively) and corroborate previous observations from this laboratory (Mitrovic and Napier, 199513; Napier e t al., 1995). Responses to the peptides were slow in onset (on average 15 to 30 sec) and often per- sisted after the termination of the ejection current (1 min or more; Fig. 1A and 2A). The effects of DAMGO and SP on the VP neurons were consistent and reproducible, and tachyphylaxis was not observed (see Fig. 3A).

Antagonism of SP responses by CP96345 I t was demonstrated previously in this laboratory

that iontophoresis of the mu opioid receptor-specific antagonist CTOP blocks the effects of the mu agonist DAMGO (Mitrovic and Napier, 1995b). This finding ver- ified that the inhibitions elicited by the opioid were evoked through a mu opioid receptor-specific action. To confirm that the effects of SP were due to selective activation of tachykinin receptors, CP96345, a NK, receptor antagonist, was used. However, this drug also is a L-type Ca2+ channel blocker (see McLean et al., 1993; Schmidt et al., 1992; for review, see Regoli et al., 1994). Thus, CP96344, an enantiomer that readily blocks Ca2+ channel but is without antagonistic properties for the NK1 receptor (McLean et al., 1993; Regoli et al., 19941,

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Fig, 1. Effects of a single ejection current of SP on neuronal responses evoked by incrementally appliedDAMG0. A: A rate histogram from one VP neuron illustrating ejected current-related response of DAMGO, and the ability of single ejection current of SP to attenuate effects of DAMGO. Arrows at the ends ofhorizontal bars indicate onset and offset of ejection current. The numbers above the bars illustrate ejection current level in nA. Vertical bars indicate 5 nA ejection current increments used to expel DAMGO. Note that after DAMGO ejection was terminated (during co-administration with SP, right side of the figure), neuronal activity returned promptly to the pre-DAMGO firing rate (i.e., SP firing rates). The activity stayed at the same level for the duration (approximately 1.5 min) of the post-DAMGO application of SP. After termination of SP ejection, firing rate returned to pretreatment activity. B: Ejection current-response curves generated for the 14 neurons tested with incremental applications of DAMGO. Data are presented as percent of control rates for the ejection currents ranging from 5 to 35 nA. Circles indicate the current-response curve obtained with DAMGO alone (pretreatment firing rates served as controls). Triangles indicate the current-response curve obtained for DAMGO when a single ejection current of SP was co-iontophoresed (rates obtained during SP single current application served as controls).

was used to decipher the contribution of Ca2+ channel blockade on any antagonism of SP by CP96345. Five VF' neurons that responded to SP were tested with both CP96345 and CP96344 using the same ejection protocol for both antagonists. In all five neurons, CP96345 abolished SP-induced neuronal excitations whereas CP96344 was ineffective (Fig. 3A,B). These results demonstrate that the SP-induced effects were elicited through the activation of NK, receptors.

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Fig. 2. Effects of a single ejection current of DAMGO on neuronal responses evoked by incrementally applied SP. A: A rate histogram from one VP neuron illustrating SP ejection current effects, and the ability of a single ejection current of DAMGO to alter neuronal responses evoked by SP applied in 5 nAincrements. The neuron depicted in this illustration was not sensitive to iontophoretically applied DAMGO, even when tested with ejection currents of up to 40 nA. However, when co-applied with DAMGO, incrementally applied SP was unable to generate a neuronal response of the magnitude that was elicited when SP was applied alone. Neuronal activity returned to the pre-SP firing rate upon termination of the SP ejection. B: Ejection current-response curves generated for seven VP neurons tested with incrementally applied SP. Data are presented as percent of control rates. Neuronal responses were analyzed for the ejection currents ranging from 5 to 35 nA. Triangles indicate current-response curve obtained with SP alone (pretreatment firing rates served as controls). Circles indicate the current-response curve obtained when a single ejection current of DAMGO was co-iontophoresed with SP (rates obtained during DAMGO single current application served as controls).

Current-dependency of neuronal responses to the peptides

The magnitude of the responses to the iontophoretic treatments was proportional to the magnitude of the ejection current used to expel the peptides as populational ejection current-response curves for DAMGO and SP (Fig. 1B and 2B, respectively) demonstrated significant linear regressions (r = 0.996, t = 37.534, P < 0.001; r = 0.973, t = 10.320, P < 0.001, respectively). Microiontophoretic application of DAMGO (ejection


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Fig. 3. Effects of the NK1 receptor antagonist CP96345, and its inactive enantiomer CP96344, on SP-elicited increases in the activity of VP neurons. A A rate histogram obtained from one I T neuron illustrating the ability of CP96345 (40 nA) to block SP-evoked increases in neuronal firing (40 nA). In contrast, CP96344 (40 nA) was unable to antagonize SP-induced effects on neuronal activity. B: A bar graph illustrating the effects of CP96345 and CP96344 on neuronal responses to SP among five VP neurons with a mean pretreatment firing rate of 9.3 5 3.6 Hz. The group of three bars on the left demon- strates the ability of CP96345 to antagonize SP-induced increases in firing. CP96344 fails to antagonize the effects of SP (three bars on the right; *P < 0.05 from pretreatment firing rates).

currents ranging from 5-35 nA) decreased neuronal activity with an Em, of 49 ? 7% below the pretreatment rates, an EcuSO of 19.1 ? 1.6 nA, and a slope of -2.81 t 0.37. Incremental application of SP produced excitations (ejection currents ranging from 5-35 nA) with a n Em,, of 54 t 13% above the pretreatment activity level, an EcuSO of 20.8 5 2.3 nA, and a slope of 2.59 ? 0.51. These pharmacologic profiles for DAMGO and SP are very similar to those previously obtained for the VP neurons using the same techniques for the drug administration and recording of neuronal activity (Mitrovic and Napier, 1995b; Napier et al., 1995, respectively).

Interaction between DAMGO and SP Twenty-three (of 63 tested) VP neurons did not re-

spond to either peptide implying that some VP neurons are not regulated by either mu-activating opioids or SP (Table I). In contrast, 20 cells responded to both DAMGO and SP (Table I) suggesting that a significant

portion of VP neurons are influenced by both opioid- and SP-containing inputs. To explore a possibility that the two peptide systems co-modulate VP neuronal activity, the ability of one peptide to alter ejection current- response curve of the other was investigated. Sixteen VF' neurons that exhibited a decrease in activity in response to the iontophoretically applied DAMGO were tested for the possibility that SP could modulate this response. Thus, DAMGO-induced effects were re-examined during co-iontophoresis of SP using single ejection current of SP ranging from 5-20 nA that produced either subthreshold effects or near-threshold effects in VF' firing (24 ? 4% change of pretreatment activity). Incremental application of DAMGO was preceded by at least 2 min single ejection current of SP (see Fig. 1A) allowing for potential effects of SP to develop and reach "steady state." To separate the effects of SP on VF' firing from its ability to modulate DAMGO-induced responses, DAMGO-evoked firing rate changes were analyzed as percent change from the activity level immediately preceding incremental application of the opioid (i.e., during recorded application of SP alone). (The same procedure was repeated for the analysis of DAMGOs effects on the ejection current-response curve of SP.) With this protocol, SP altered the profile of the neuronal responses to the mu opioid agonist in 14 of 16 neurons (Fig. 1B). Four of these 14 neurons did not meet criteria for the sensitivity to SP (i.e., a change of firing rate by a t least 20% using ejection current level of 30 nA). Nevertheless, SP (20 nA) was able to attenuate DAMGO-induced inhibition of neuronal activity with each of these four neurons. Co-iontophoresed SP reduced DAMGOs Em, from 49 ? 7% to 22 ? 6% below the baseline firing (t = -7.001; P < 0.051, the slope from -2.81 ? 0.37 to -1.44 t 0.44 (t = -4.473; P < 0.051, and increased the EcuSO from 19.1 ? 1.57 nA

Eight neurons of the 31 that increased firing rate in response to the iontophoretically applied SP were tested to determine if DAMGO could modulate these neuronal responses. DAMGO was expelled from the pipette using ejection currents ranging from 15-40 nA which reduced firing by 12 ? 6% of pretreatment activity. Using this protocol, DAMGO attenuated the effects of SP in seven of the eight tested neurons (Fig. 2B). The Em, was re- ducedfrom 53.9 ? 13.4% to 19.3 ? 7.3% above the baseline firing (t = 2.885; P < 0.051, and the slope from 2.59 ? 0.51 to 1.15 ? 0.19 (t = 3.382; P < 0.05). The EcuSO obtained for SP during co-iontophoresis with DAMGO equaled 22.0 ? 1.64 nA. This was not different (t = -0.466; P = n.s.1 from the one obtained when SP was applied alone (20.8 ? 2.3 nA). Four neurons (out of these seven) demonstrating SP-induced excitations did not fulfill the criteria for sensitivity to DAMGO (20% change by 30 nA). However, DAMGO co-iontophoresis (20-40 nA) attenuated excitations elicited by SP in each of these neurons (see Fig. 2A).

to 23.93 -+- 1.66 nA (t = -3.337; P < 0.05).

148 I. MITROVIC AND T.C. NAPTRR

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Fig. 4. Recording site location for neurons tested with DAMGO and SP. Filled circles represent neurons that responded to iontophoretically applied DAMGO, and filled triangles indicate neurons that responded to SP. Filled squares locate the neurons that responded to both DAMGO and SP. Neurons not responding to either DAMGO, SP or both are represented with open circles, triangles, and squares, respectively.

Histology Reconstruction of the recording sites demonstrated

that the neurons sampled in the present study were located throughout rostro-caudal, medio-lateral, and dorso-ventral extent of the VP and the sublenticular substantia innominata (Fig. 4). The topographic distribution of the recorded neurons was analyzed as a function of the neuronal responses to DAMGO, SP and both peptides. Corroborating previously published results (Mitrovic and Napier, 1995b; Napier et al., 1995) the ratio of sensitive vs. non-nonsensitive neurons for each of the peptides was comparable throughout the eight subregions of the VP and adjacent substantia innominata suggesting homogeneous innervation of the region by both enkephalins and SP. A homogenous distribution also was observed for the subpopulation of neurons that demonstrated sensitivity to both neuropeptides.

DISCUSSION Analysis of the electrophysiological characteristics of

the recorded neurons, the neuronal responses to DAMGO and SP, and the anatomical location of the

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Numbers in upper right corner of each drawing indicate distances (in mm) from bregma (Paxinos and Watson, 1986). Abbreviations: ac, anterior commissure; FStr, fundus striati; GP, globus pallidus; HDB, horizontal limb of diagonal band; MCPO, magnocellular preoptic nucleus; SI, substantia innominata; VP, ventral pallidum.

recording sites indicate that the population of VP neurons tested was similar to that previously investigated with these peptides (Mitrovic and Napier, 1995b; Napier e t al., 1995). Using the mu opioid-specific antagonist CTOP, Mitrovic and Napier (1995b) demonstrated a mu opioid receptor-selective action by iontophoretically applied DAMGO. In the present study, the NK1 receptor- specific antagonist CP96345 blocked the effects of SP demonstrating a NK, tachykinin receptor-specific action of SP. Because CP96345 also is a Ca2' channel blocker (Schmidt et al., 19921, the lack of antagonism by its enantiomer CP96344 (a Ca2+ blocker which has no biological activity at tachykinin receptors; McLean et al., 1993; also, see Regoli et al., 1994) confirmed that the SP-induced effects were mediated through the NK, receptor.

In agreement with previous studies (Mitrovic and Napier, 1995; Napier et al., 19951, DAMGO decreased VP neuronal firing and SP induced rate increases. These observations also concur with work in other brain regions where SP and opioids have been characterized excitatory and inhibitory neuropeptides, respectively


(e.g., Nakajima et al., 1991; North, 1993, respectively). Because the two peptides exerted oppositional effects through the activation of different receptors (mu and NKJ it is not surprising that their interaction in the regulation of VP activity demonstrated attributes of a functional antagonism. Near-threshold ejection currents of SP decreased Em,, and slope, and increased EcuSO of ejection current-response curve generated for DAMGO. Also, near-threshold ejection currents of DAMGO attenuated the effects of SP (without changing the EcuSO). Similar modulations occurred in some neurons that responded to iontophoretic application of only one of the peptides. When the peptide that did not produce a response was applied as the modulator, it readily altered ejection current-response curve of the other peptide (i.e., principal agonist). A plausible explanation for the observed phenomenon may be that the modulating peptide did not achieve concentrations sufficient to activate the number of receptors necessary to evoke changes in neuronal activity, but it did activate an ade- quate number of receptors to precipitate effects that are subthreshold to generation of action potentials. Because the present study evaluated firing rate changes (i.e., changes in the number of action potentials occurring over time), these results suggest that the mechanisms underlying the functional antagonism of opioids and tachykinins occur at that subthreshold level.

Several ultrastructural arrangements would result in the peptide interactions observed in the present study. One possibility assumes a presynaptic localization for NK, and mu receptors, both controlling the release of an excitatory neurotransmitter. The release of this transmitter might be stimulated through the activation of the NK1 receptor and inhibited through the activation of the mu receptor. There is evidence for a presynaptic localization of these receptors (Glowinski et al., 1993; Goodman et al., 1988). Also, opioids can inhibit and SP can stimulate the release of excitatory amino acids within the central nervous system (e.g., Simmons et al., 1994; Skilling and Larson, 1993). Gluta- mate increases VP neuronal firing (Lamour et al., 1986; Napier et al., 1991). A major source of glutamatergic inputs is the basolateral amygdala (Carnes et al., 1990; Fuller et al., 1987), and electrical stimulation ofthe basolateral amygdala can induce a short-onset excitation of VP firing consistent with a monosynaptically mediated effect (Maslowski-Cobuzzi and Napier, 1994; Yim and Mogenson, 1983). Recent work has demonstrated that this evoked excitation is altered by both DAMGO and SP (Mitrovic and Napier, 1995a). Surprisingly however, DAMGO potentiated amygdala-evoked excitations in the VP, and SP attenuated the response. If projections from the amygdala are prototypical of VP glutamatergic inputs (which also include those from the cortex and sub- thalamic nucleus), then such results argue against a di- rect, presynaptic control on the release of glutamate by either mu opioid or NK1 tachykinin receptors.

Another morphologic arrangement that would ex- plain the neuropeptide interactions observed in the present study assumes a postsynaptic location for both mu and NK, receptors. Under this circumstance, mu- tual regulation could take place through the activation of receptors co-expressed by the recorded neuron. A number of studies have demonstrated postsynaptic localization of n u and NKI receptors (e.g., Fallon and Leslie, 1986; Liu et al., 1994). Both the mu and NK1 receptors are known to be coupled to G-proteins (for review, see Evans, 1993; Krause et al., 1994), and the activation of these receptors results in altered ionic conductances (Nakajima and Nakajima, 1994; North, 1993). Thus, it is possible that one peptide could decrease the efficiency of coupling within the signal transduction cascade of the other, resulting in a functional antagonism with altered slope (and EcuSO) like that observed in the present study. This phenomenon was observed in cultured locus coeruleus neurons where SP and met-enkephalin invoke (G-protein mediated) op- posing effects in the regulation of an inwardly rectifying K' channel (Velimirovic et al., 1995). Met-enkephalin opens, and SP closes this channel. Thus, co-regulation of a common conductance is a way by which these two peptide systems could oppositionally regulate the function of a VP cell.

It is noteworthy that not all encountered VP neurons were influenced by both DAMGO and SP. Twenty VP neurons (out of 63) responded to only one iontophoretically applied peptide. Nine of these responded only to iontophoretically applied DAMGO, and 11 to SP. These data indicate that there are significant populations of VP neurons that are regulated by either opioids or SP, but not both.

The VP is involved in the regulation of several behaviors, including locomotion (see Mogenson and Yang, 19911, cognition (see Olton et al., 1991) and reward (e.g., Hubner and Koob, 1990). In light of these functions, the interaction between mu receptor-activating opioids and SP may have a profound behavioral relevance. The motoric effects of opioid and SP agonists microinjected within VP have been described (Austin and Kalivas, 1990; Hoffman et al., 1991; Napier, 1992; Napier et al., 1995). In an apparent discord with the present results, both intra-VP injections of DAMGO (Austin and Kali- vas, 1990; Hoffman et al., 1991; Napier, 1992), and relatively high doses of a peptidase resistant tachykinin DiMeC7 (Napier et al., 1995) increase motor behavior. However, it is possible that the VP mu opioid and NK1 receptors whose activation alters motoric function do not interact in the regulation of neuronal activity. The finding that 31% of the VP neurons tested responded to only one iontophoretically applied peptide supports this possibility.

It also is thought that the VP is involved in the mediation of hedonic effects of abused drugs (e.g., Hubner and Koob, 1990). However, intra-VP microinjections of

150 I. MITROVIC AND T.C. NAPIER

DAMGO (Johnson et al., 1993) or SP (caudal W; Holz- hauer-Oitzl et al., 1988) have reinforcing effects suggesting that the observed oppositional interaction may not have a significant role in regulating reinforcement.

Cognitive functions have been associated with VP cholinergic neurons (see Olton et al., 1991). These neurons project to the frontal cortex and basolateral amygdala (Mesulam et al., 1983; Saper, 1984). Microinjection of opioid agonists into VP decreases cholinergic trans- mission in both frontal cortex and basolateral amygdala (Napier et al., 1993). Additionally, microinjections of the SP in the caudal VP facilitates learning in an inhibitory avoidance task (Gerhardt e t al., 1992; Kafetzopoulos et al., 1986). On the contrary, cognitive functions are impaired following central or peripheral administration of opioids (e.g., Gallagher e t al., 1987; Aloyo et al., 1993, respectively). Thus, it is tempting to speculate that the mu-activating opioids may interact oppositionally with SP to regulate cognitive functions within the VP.

The aforementioned behaviors are not isolated from each other. Behavioral studies, as well as patterns of neuronal connectivity within the basal forebrain, suggest that the integration of VP pathways is important for complex behaviors that are critical for the survival of an animal. For example, the recognition of significant environmental circumstances, the assignment of af- fective value to these, and execution of an appropriate motoric reaction are complex behavioral sequences that necessitate an orchestrated activation of basal forebrain circuity. These “goal-directed behaviors” require cognition, reward, and motoric behavioral modalities (see Mogenson and Yang, 19911, all known to be within the functional repertoire of the VP. Because opioids and tachykinins modulate these behaviors, studying their interaction in the VP should provide an important in- sight for understanding the normal basal forebrain physiology, as well as the neuropathologies that are characterized by dysfunctions in motivated behavior such as schizophrenia, mania, and depression.

ACKNOWLEDGMENTS The authors extend their gratitude to Drs. P.I. John-

son, M. Margeta-Mitrovic, and B.S. Heidenreich for their valuable comments. We also are very grateful to Pfizer, Inc. for the generous gifts of CP96345 and CP96344. This work was supported by USPHSG DA 05255 to T.C.N.

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Interactions between the mu opioid agonist DAMGO and substance P in regulation of the ventral pallidum