DNQX

A-Opioids disinhibit and n-opioids inhibit serotonin efflux in the dorsal raphe nucleus

Abstract

The relative importance of GABAergic and glutamatergic afferents in mediating the effects of A- and n-opioids on serotonin (5-HT) efflux in vivo has not been firmly established. Thus, we used microdialysis in the dorsal raphe nucleus (DRN) of freely behaving rats to study the effect of GABA and glutamate receptor antagonists on opioid-induced changes in 5-HT efflux. Infusing the A-opioid agonist DAMGO (300 AM) increased extracellular 5-HT in the DRN by ¨70%. During infusion of the GABAA receptor blocker bicuculline (100 AM), extracellular 5-HT increased by ¨250%, and subsequent infusion of DAMGO decreased 5-HT to ¨70% above the pre-bicuculline baseline. These data are consistent with the hypothesis that A-opioids disinhibit 5-HT neurons, an effect attenuated by direct inhibition of 5-HT efflux or inhibition of excitatory influences on 5-HT efflux. To further test this hypothesis, glutamate receptor blockers, AP-5 (1 mM) and DNQX (300 AM), were co-infused with DAMGO. The glutamate receptor antagonists prevented decreases in 5-HT elicited by DAMGO in the presence of bicuculline. This indicates that DAMGO inhibits glutamatergic afferents, which partly offsets the disinhibitory influence of A-opioids on 5- HT efflux. In contrast, the n-opioid agonist, U-50,488 (300 AM), decreased 5-HT by ¨30% in the DRN. Glutamate and GABA receptor antagonists did not block this effect. In conclusion, A-opioids inhibit GABAergic and glutamatergic afferents, thereby indirectly affecting 5- HT efflux in the DRN.
In contrast, n-opioids inhibit 5-HT efflux independent of effects on glutamatergic and GABAergic afferents.

Keywords: Serotonin; GABA; Glutamate; Opioid; Dorsal raphe nucleus; Microdialysis

1. Introduction

In the dorsal raphe nucleus (DRN), neurons containing serotonin (5-HT) regulate behavioral state [7] and may play a role in responses to stress [22]. According to some reports, opioid-induced 5-HT efflux may increase pain threshold, and this could be beneficial in coping with stress [1,24]. However, the mechanisms by which opioids influence 5-HT neurons have not been fully established. Opioid-containing synaptic endings are present in the DRN [35], but opioid receptor agonists do not directly excite 5-HT neurons [9]. Instead, the stimulatory influence of opioids on 5-HT efflux may be indirectly mediated. GABAergic neurons in the rat DRN express A-opioid receptors [11] and have a tonic inhibitory influence on 5-HT neurons [4,29]. In vitro administration of A-opioids suppressed inhibitory postsy- naptic currents (IPSCs) recorded from presumptive 5-HT neurons in the DRN [9]. Consistent with this result, 5-HT efflux increased in response to morphine, an effect that was attenuated by pretreatment with GABAA receptor agonists and antagonists [25,26]. Together, these studies suggest that A-opioids inhibit GABAergic afferents and thus disinhibit 5- HT neurons in the DRN.

Glutamatergic neurons may also play a role in opioid effects on 5-HT neurons. Thus, similar to the effect on IPSCs, A-opioids suppressed excitatory postsynaptic cur- rents (EPSCs) recorded from 5-HT neurons in the DRN [9]. However, glutamate receptor antagonists attenuated mor- phine-induced increases in 5-HT efflux in vivo [26], suggesting that opioids enhance rather than restrain excita- tory synaptic input. Possibly, by reducing GABA tone in the DRN, opioids potentiate the excitatory influence of gluta- mate on 5-HT neurons, an effect offset partly by direct inhibition of glutamatergic afferents. These observations suggest that the net effect of A-opioids depends on the initial balance between GABAergic and glutamatergic influences on 5-HT neurons.

n-Opioids inhibit 5-HT efflux [27]. This might involve effects on GABAergic and glutamatergic synaptic trans- mission. Thus, evoked EPSCs in the DRN were inhibited by n-opioids [20], and some GABA-containing neurons in the medulla express n-opioid receptors [12]. However, the importance of indirect compared to direct n-opioid effects on 5-HT efflux has not been determined. To further investigate the role of afferents in mediating opioid effects on 5-HT neurons, we used in vivo microdialysis in the DRN of freely behaving rats. Thus, opioid-induced changes in 5- HT efflux can be determined when afferents are intact and compared to effects of opioids after GABA and glutamate receptors are blocked. In the present study, we compared the effect of infusing the selective A-opioid receptor agonist DAMGO and the selective n-opioid receptor agonist U- 50,488 in the DRN. Bicuculline was infused into the DRN to block GABAA receptors. AP-5 combined with DNQX was infused to block ionotropic glutamate receptors.

2. Materials and methods
2.1. Animals

Male Sprague– Dawley rats (Harlan Sprague– Dawley Inc., Indianapolis, IN) were individually housed with food and water available ad libitum. The animals were kept on a reversed light– dark cycle (lights-off: 9:30 – 21:30), and all experiments were performed during the lights-off period. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Rutgers University Institutional Review Board. Rats weighing 300 – 350 g were anesthetized with a combination of xylazine (4 mg/kg, i.p.) and ketamine (80 mg/kg, i.p.), and guide cannulas were implanted above the targets. For the DRN guide cannulas, the coordinates were: anterior– posterior 1.2 mm relative to interaural zero;medial– lateral 4.0 mm at a 32- angle lateral to midline; dorsal– ventral 2.0 mm below the surface of the skull [19]. For Fdual probe_ experiments, guide cannulas were implanted in the nucleus accumbens (NAcc) in addition to the DRN. The coordinates for the guide cannulas in the NAcc were: anterior– posterior 10.7 mm relative to inter- aural zero, medial– lateral 1.4 mm; and dorsal– ventral 2.0 mm below the skull surface. After implantation, the guide cannulas were plugged with obdurators, and the animals were allowed a recovery period of at least 1 week.

2.2. Microdialysis

Microdialysis was performed with an I-shaped probe constructed from 26-gauge stainless steel tubing and glass silica. The dialysis tubing was hollow nitrocellulose fiber (200 Am i.d., 13,000 MW cut-off; Spectrum Medical Industries, Los Angeles, CA). The length of the dialysis membrane exchange surface was 1.0 mm for the DRN and 2.5 mm for the NAcc. The probe was treated with 70% alcohol immediately before implantation.

The evening before an experiment, rats were briefly anesthetized with ether, and aseptic dialysis probes were inserted via the guide cannulas and secured with dental cement. The coordinates for the tip of the microdialysis probes in the DRN were: medial– lateral T1.0 mm at a 32- angle to the midline; anterior– posterior 1.2 mm; dorsal– ventral 6.4 mm. In the NAcc, the coordinates were: anterior– posterior 10.7 mm, medial– lateral 1.4 mm and dorsal– ventral 8.5 mm. Rats were then placed in the test chamber with food and water available and attached to a fluid swivel that allowed relatively free movement. The dialysis probes were perfused overnight with a modified, buffered Ringer solution (140 mM NaCl, 3.0 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 0.27 mM NaH2PO4, 1.2 mM Na2HPO4, 1 AM citalopram, pH 7.4). This Ringer solution (artificial cerebrospinal fluid; aCSF) was pumped at a rate of 1.0 Al/min. Sample collection began the following morning at the beginning of the lights-off period under dim red light conditions.

The selective 5-HT reuptake blocker citalopram was included in the aCSF to increase the reliability of 5-HT detection. Citalopram at 1 AM in the aCSF is at the low end of the dose– response curve for increasing extracellular 5- HT in the DRN and does not strongly activate autoreceptors or inhibit 5-HT release under our experimental conditions [31]. Release was measurably inhibited only when citalo- pram was included at concentrations greater than 10 AM [31]. The concentration of infused substances in extracel- lular space falls exponentially as a function of distance from the dialysis probe [3]. This suggests that citalopram elevates extracellular 5-HT only in a small area adjacent to the microdialysis probe and autoreceptors are not sufficiently activated to inhibit 5-HT neuronal activity. Moreover, citalopram did not qualitatively alter the effects of exper- imental manipulations such as blocking GABAA and glutamate receptors or systemic administration of opioids. For example, local infusion of bicuculline into the DRN produced a 3-fold increase in extracellular 5-HT irrespective of the presence of a reuptake inhibitor [28].

Samples were collected every 30 min and analyzed by high performance liquid chromatography with electrochem- ical detection. Separation of 5-HT was achieved on a 10 cm × 3.2 mm column with ODS 3 Am packing (BAS Inc., W.Lafayette, IN). The mobile phase composition was 0.12 M NaOH, 0.18 mM EDTA, 0.15 M monochloroacetic acid, 1.0 mM sodium octane sulfonic acid and 56 ml/l acetonitrile, pH 3.4, and was pumped at a rate of 0.90 ml/min. Monoamines were measured using a dual potentiostat electrochemical detector (EG&G PARC, Oak Ridge, TN) and dual glassy carbon electrode electrodes in the parallel configuration. Applied potentials, relative to an Ag/AgCl electrode, were set at approximately maximal and half-maximal values (550 and 490 mV) for oxidation of 5-HT. The detection limit for 5-HT was approximately 0.3 pg per sample based on a signal-to- noise ratio of 3:1.

2.3. Experimental design and data analysis

Mean baseline 5-HT levels were calculated as the average of the four successive samples before drug administration and reported in the figure legends as pg/sample, uncorrected for probe recovery. Furthermore, the data were normalized and presented in figures as mean T SEM percent change from the averaged baseline measurements. Significance ( P < 0.05) was determined using repeated measures ANOVA followed by post-hoc Scheffe`’s test. To determine if afferents mediate the effects of opioids on 5-HT, rats were pretreated with GABAergic or glutamatergic receptor antagonists before and during administration of the A- agonist DAMGO and the n-agonist U-50,488. All drugs were administered by reverse dialysis infusion into the DRN. We used bicuculline to block GABAA receptors and a combination of AP-5 and DNQX to block ionotropic glutamate receptors as previously described in detail [29,30]. To evaluate the effects of bicuculline and AP-5 + DNQX or the combination, 5-HT levels were expressed as a percent change from baseline before infusing DAMGO and U-50,488. Because the time and duration to reach pro- nounced effects were different, data for DAMGO and U- 50,488 were calculated differently. For DAMGO, the maximum effect was observed in the 3rd and 4th samples after administration. Thus, the percent change was obtained by dividing the average of these two samples by the average of two consecutive samples immediately before DAMGO administration. For U-50,488, the maximum decrease was observed in the 3rd through 6th samples after administra- tion. The percent change was obtained by dividing the average of these four samples by four consecutive samples immediately before U-50,488. Unpaired Student’s t test ( P < 0.05) was used to compare the mean values shown in bar graphs (Figs. 4C and 6B).Bicuculline methiodide, (T)-2-amino-5-phosphonopenta- noic acid (AP-5) and 6,7-dinitro-quinoxaline-2,3-dione (DNQX) were purchased from Sigma-RBI (Natick, MA). DAMGO and U-50,488 were purchased from Sigma (St. Louis, MO). 3. Results 3.1. Effect of opioid receptor agonists on 5-HT efflux in the DRN After obtaining stable baseline measurements of extracellular 5-HT in the DRN, DAMGO (300 AM) or U-50,488 (300 AM) was infused by reverse dialysis for 2 h in the DRN. The concentration of these opioid receptor agonists in the aCSF was based on dose– response experiments and the ability of selective A- and n-opioid receptor antagonists to block the changes in extracellular 5-HT induced by infusion of DAMGO and U-50,488 into the DRN [27]. As shown in Fig. 1, DAMGO (300 AM) increased extracellular 5-HT in the DRN. The maximum increase was 76.7% T 16.3% above the mean baseline level. Extracellular 5-HT gradually returned towards baseline levels after the end of DAMGO infusion. In contrast, U-50,488 (300 AM) decreased extracellular 5-HT in the DRN. The maximum reduction was 27.5% T 3.9% (Fig. 1). 3.2. Effect of a GABAA receptor antagonist on l-opioid-induced increases in 5-HT efflux DAMGO inhibited GABA-mediated IPSCs recorded from 5-HT neurons in the DRN [9], supporting the conclusion that A-opioid-induced increases in 5-HT efflux result from disinhibition of 5-HT neuronal activity. To further test this hypothesis, the GABAA receptor antagonist bicuculline (100 AM) was infused into the DRN with the addition of DAMGO (300 AM) 3 h after the start of bicuculline. As shown in Fig. 2, during bicuculline infusion alone, extracellular 5-HT in the DRN increased ¨3-fold. Then, during the following 2 h period of combined DAMGO and bicuculline infusion, extracellular 5-HT decreased significantly to ¨170% of the original baseline level (Figs. 2, 4C). Thus, a GABAA receptor antagonist prevented the DAMGO-induced increase in 5-HT efflux. Instead, relative to the elevated levels during the previous period of bicuculline infusion, DAMGO induced a decrease in 5-HT efflux (Fig. 4C). Despite the significant decrease in efflux, extracellular 5-HT remained elevated relative to the original pre-bicuculline baseline. 3.3. Effect of glutamatergic receptor antagonists on l-opioid-induced increases in 5-HT efflux To test the role of endogenous glutamate in opioid- induced changes in extracellular 5-HT, we infused AP-5 and DNQX into the DRN to block NMDA and non-NMDA ionotropic glutamate receptors. As shown in Fig. 3, combined infusion of AP-5 (1 mM) and DNQX (300 AM) decreased extracellular 5-HT by ¨20 – 30% below baseline levels in the DRN. During infusion of AP-5 and DNQX, DAMGO (300 AM) still produced an increase in 5-HT (Fig. 3). However, as normalized to 5-HT levels just before DAMGO infusion, the response with glutamate receptors blocked was significantly smaller than the effect of DAMGO alone (Fig. 4C). Fig. 2. Effect of the GABAA receptor antagonist bicuculline on DAMGO- induced changes in DRN 5-HT. The data are expressed as mean (TSEM) percent change from the pre-drug baseline levels. The open and hatched horizontal bars indicate the period of respectively, bicuculline and DAMGO infusion into the DRN. During bicuculline infusion, DRN 5-HT was increased by 7.3 T 1.9 pg from the baseline of 4.0 T 1.0 pg. During bicuculline infusion, DAMGO produced a significant reduction in DRN 5- HT [F(1,16) = 11.488, P = 0.0037]. The decrease in 5-HT was 4.9 T 1.5 pg from the pre-DAMGO level of 11.5 T 2.9 pg. *P < 0.05. Fig. 3. Effect of infusion of glutamate receptor antagonists on DAMGO- induced increases in 5-HT. The data are expressed as mean (TSEM) percent change from the pre-drug baseline levels. The open horizontal bar indicates infusion into the DRN of AP-5 (1000 AM) + DNQX (300 AM), and the solid bar is the period of DAMGO (300 AM) infusion into the DRN. The mean baseline level of extracellular 5-HT was 6.8 T 1.4 pg/sample (n = 18). As compared to the AP-5 and DNQX control, DAMGO produced a significant increase in DRN 5-HT [F(1,16) = 5.386, P = 0.0338]. *P < 0.05. 3.4. Effect of combined infusion of GABA and glutamate receptor antagonists on l-opioid-induced increases in 5-HT efflux A-Opioids also inhibit excitatory afferents to 5-HT neurons in the DRN, an effect observed in vitro when GABAA receptors were blocked [9]. This is one possible explanation for the decrease in 5-HT efflux produced by DAMGO during bicuculline infusion (see Fig. 2). To study the role of endogenous glutamate in DAMGO- induced decreases in 5-HT, we infused bicuculline followed by AP-5 and DNQX. Similar to previous experiments, bicuculline (100 AM) infusion into the DRN increased extracellular 5-HT ¨two- to three-fold. Subsequent infusion of AP-5 (1 mM) and DNQX (300 AM) reduced 5-HT to ¨140% of the initial baseline (Fig. 4A). In the presence of GABAA and glutamate receptor antagonists, infusion of DAMGO (300 AM) into the DRN neither decreased nor increased 5-HT. Although there was a tendency towards an increase in 5-HT, this difference was not significant compared to the control group ( P > 0.05). One explanation for the tendency towards an increase in 5-HT is the possibility that GABAA receptors were not completely blocked by bicuculline at a concentration of 100 AM in the aCSF. To test this hypothesis, we replicated this experiment, with the bicuculline concentration in the aCSF increased to 300 AM. As shown in Figs. 4B and C, 5-HT increased ¨three-fold during infusion of 300 AM bicuculline and then decreased to ¨175% of the initial baseline during infusion of AP-5 combined with DNQX. Subsequent infusion of DAMGO produced no further change in 5-HT (Figs. 4B and C). In summary, 5-HT efflux did not significantly change when DAMGO was added after a period of combined blockade of GABAA and glutamate receptors. In contrast, relative to levels just prior to DAMGO, 5-HT efflux decreased when the A-opioid was added after infusion of bicuculline alone, while 5-HT efflux increased in response to DAMGO alone. Although the effects on efflux differed among the treatment groups (Fig. 4C), extracellular 5-HT during DAMGO infusion reached ¨170% of the initial baseline in all cases, and this was similar to the effect of combined infusion of the GABAA and glutamate receptor antagonists without DAMGO.

3.5. Effect of GABAA and glutamate receptor antagonists on j-opioid-induced decreases in 5-HT efflux

To test the role of GABAergic and glutamatergic afferents in mediating the effects of a n-opioid, U-50,488 was infused into the DRN after first blocking GABAA or ionotropic glutamate receptors. During infusion of bicucul- line (100 AM) into the DRN, 5-HT increased to ¨300% of baseline (Fig. 5A). Subsequent infusion of U-50,488 (300 AM) into the DRN reduced extracellular 5-HT to ¨160% of initial baseline levels in the DRN. Based on the decrease from normalized baseline 5-HT levels, bicuculline signifi- cantly enhanced the inhibitory effect of U-50,488 on extracellular 5-HT in the DRN. As a percentage of normalized baseline levels, the decrease was ¨47% in the presence of bicuculline compared to ¨27% without the GABAA receptor antagonist (Fig. 6B).

We next tested the possibility that the effect of U-50,488 on 5-HT was indirectly mediated by inhibition of gluta- matergic afferents. Consistent with previous results, com- bined infusion of the glutamate receptor antagonists AP-5 (1 mM) and DNQX (300 AM) produced a small, ¨20% reduction in DRN 5-HT (Fig. 5B). In the presence of glutamate receptor antagonists, U-50,488 (300 AM) infusion into the DRN induced a further reduction in extracellular 5- HT to ¨50% below baseline level in the DRN (Fig. 5B). Based on the decrease from normalized baseline 5-HT levels, the glutamate receptor antagonists did not block but significantly enhanced the inhibitory effect of U-50,488 on extracellular 5-HT in the DRN. Thus, the mean decrease in 5-HT was ¨41% from the normalized pre-drug baseline level compared to ¨27% without the glutamate receptor antagonists (Fig. 6B).

Fig. 4. Effect of combined infusion of GABAA and glutamate receptor antagonists on DAMGO-induced changes in DRN 5-HT. The data are expressed as mean (TSEM) percent change from the pre-drug baseline levels. The open horizontal bar indicates infusion into the DRN of bicuculline, and the hatched bar indicates infusion into the DRN of AP-5 (1000 AM) + DNQX (300 AM). The solid bar indicates the period of DAMGO (300 AM) infusion into the DRN. (A) The mean baseline level of extracellular 5-HT was 5.5 T 1.0 pg/sample (n = 13). Bicuculline (100 AM), AP-5 (1 mM) and DNQX (300 AM) were infused into the DRN before and during DAMGO. With this treatment, there was a tendency towards an increase in 5-HT during DAMGO infusion. However, this effect was not significant compared to the control group [F(1,11) = 1.864, P = 0.1995]. (B) Bicuculline (300 AM), AP-5 (1 mM) and DNQX (300 AM) were infused into the DRN before and during DAMGO. The mean baseline level of extracellular 5-HT was 3.3 T 0.3 pg/sample (n = 5). Pretreatment with the high concentration of bicuculline in combination with AP-5 and DNQX completely prevented DAMGO-induced increases in 5-HT. (C) The bar graph compares changes in 5-HT efflux in response to DAMGO alone, the combination of bicuculline with AP-5 + DNQX and DAMGO in the presence of bicuculline and/or AP-5 + DNQX, as described in Materials and methods. The values summarize data from Figs. 1 – 3 and panel (B). The effect of DAMGO on 5-HT was reversed by bicuculline, attenuated by AP-5 and DNQX and completely blocked by combined infusion of bicuculline, AP-5 and DNQX. *P < 0.05 vs. DAMGO alone; #P < 0.05 vs. AP-5 + DNQX and DAMGO. Fig. 5. U-50,488-induced decreases in DRN 5-HT were not attenuated by GABAA or glutamate receptor antagonists. (A) Effect of bicuculline on U-50,488- induced changes in DRN 5-HT. The data are expressed as mean (TSEM) percent change from the pre-drug baseline levels of 3.7 T 1.0 pg/sample in the experimental group and 2.8 T 1.9 pg/sample in the control group. The open horizontal bar indicates the infusion period for bicuculline (100 AM), and the hatched horizontal bar indicates infusion of U-50,488 (300 AM) into the DRN. During bicuculline infusion, U-50,488 significantly decreased 5-HT in the DRN [F(1,10) = 5.410, P = 0.0423]. In response to U-50,488, the absolute decrease in 5-HT was 3.2 T 1.1 pg from the pre-U-50,488 level of 8.3 T 1.8 pg. (B) Effect of combined infusion of AP-5 and DNQX on U-50,488-induced changes in DRN 5-HT. The data are expressed as mean (TSEM) percent change from the pre-drug baseline levels of 4.2 T 0.6 pg/sample in the experimental group and 3.7 T 1.0 pg/sample in the control group. The open and hatched horizontal bars indicate the infusion period of the glutamate receptor antagonists and U-50,488, respectively. During infusion of the glutamate receptor antagonists, U-50,488 induced a significant decrease in 5-HT [F(1,15) = 7.336, P = 0.0162]. In response to U-50,488, the absolute decrease in 5-HT was 1.1 T 0.4 pg from the pre-U-50,488 level of 3.4 T 0.7 pg. *P < 0.05. Fig. 6 shows that combined blockade of GABAA and glutamate receptors did not attenuate the inhibitory effect of U-50,488 on extracellular 5-HT in the DRN. Similar to previous experiments, bicuculline (100 AM) infusion into the DRN produced an increase in 5-HT to ¨300% of baseline levels in the DRN. Subsequent infusion of a combination of AP-5 (1 mM) and DNQX (300 AM) reduced 5-HT to ¨160% of the initial baseline. In the presence of GABAA and glutamate receptor antagonists, U-50,488 (300 AM) infusion into the DRN significantly reduced extrac- ellular 5-HT in the DRN (Fig. 6A). The effect on 5-HT, an ¨31% decrease from the normalized pre-drug baseline level, was not statistically different from the ¨27% decrease in the absence of receptor antagonists. Fig. 6. Combined infusion of GABAA and glutamate receptor antagonists did not block U-50,488-induced decreases in DRN 5-HT. (A) The data are expressed as mean (TSEM) percent change from the pre-drug baseline level of 4.5 T 0.9 pg/sample. The open and hatched horizontal bars indicate the infusion period for respectively, bicuculline (100 AM) and the combination of AP-5 (1 mM) and DNQX (300 AM). The solid bar indicates the infusion period for U-50,488 (300 AM). Infusion of bicuculline alone induced an increase of 6.2 T 1.8 pg from baseline. Subsequent infusion of AP-5 and DNQX reduced 5-HT 4.3 T 1.5 pg from the initial level of 10.7 T 2.5 pg in the presence of bicuculline. U-50,488 infusion into the DRN induced a further 1.9 T 0.6 pg reduction from the pre-U-50,488 level of 6.4 T 1.2 pg. [ F(1,1) = 16.745, P = 0.0001; *P < 0.05 vs. control]. (B) The bar graph shows changes in 5-HT in response to U-50,488 alone compared to the effect of U-50,488 in the presence of bicuculline and/or AP-5 + DNQX, as described in Materials and methods. The values summarize data from Fig. 5 and this figure. The effect of U-50,488 was significantly enhanced by pre-infusion of bicuculline (t13 = 2.902, P = 0.0124) and AP-5 + DNQX (t16 = 2.826, P = 0.0122). Combined infusion of the GABAA and glutamate receptor antagonists did not significantly alter the effect of U-50,488 (t14 = 0.852, P = 0.4066). *P < 0.05 unpaired Student’s t test. Fig. 7. Effect of infusion of U-50,488 into the DRN on 5-HT in the NAcc. The open horizontal bars indicate the time of U-50,488 infusion into the DRN. The data are expressed as mean (TSEM) percent change from the pre- drug baseline level of 2.9 T 0.3 pg/sample in the NAcc. Compared to aCSF control, U-50488 (300 – 1000 AM) produced significant decreases in the NAcc (F(1,10) = 11.497, P = 0.0069). *P < 0.05, post-hoc Scheffe´’s test. Decreased 5-HT efflux in the DRN in response to U- 50,488 infusion might result from inhibition of 5-HT neuronal activity. To test this hypothesis, we infused U- 50,488 into the DRN and measured changes in extrac- ellular 5-HT with a second microdialysis probe in the NAcc. As shown in Fig. 7, infusion of U-50,488 into the DRN produced dose-related decreases in extracellular 5- HT in the NAcc. In response to U-50,488 at a concen- tration of 300 AM in the aCSF, 5-HT decreased to ¨80% of baseline levels in the NAcc. At a concentration of 1000 AM, U-50,488 infusion decreased 5-HT to ¨60% of baseline levels in the NAcc. 4. Discussion These results support the hypothesis that A-opioids inhibit GABAergic synaptic input and thus disinhibit 5- HT neurons in the rat DRN. A-Opioids also inhibit glutamatergic afferents [9]. However, the balance between the changes in inhibitory and excitatory influences favors increased 5-HT efflux. In contrast, n-opioids reduced 5-HT efflux independent of effects on glutamatergic and GABAergic afferents to the DRN. In support of this conclusion, glutamate and GABA receptor antagonists did not block n-opioid-induced decreases in 5-HT efflux. 4.1. Interactions between l-opioids, GABA and 5-HT DAMGO infusion into the DRN increased extracellular 5-HT in the DRN. This is consistent with our report that DAMGO dose-dependently increased 5-HT efflux [27]. We observed near maximal increases in 5-HT during DAMGO infusion at a concentration of 300 AM in the aCSF, an effect that was blocked by the selective A-opioid receptor antagonist h-funaltrexamine [27]. Moreover, DAMGO infusion into the DRN also elicited 5-HT efflux in the NAcc [27]. These results suggest that the effect of DAMGO on 5-HT efflux in the DRN provides an indication of A-opioid-receptor-mediated changes in 5-HT neuronal activity and release in forebrain projection sites. The effective concentration range of DAMGO and other receptor ligands used in these experiments appears high. However, this is generally consistent with other reports using reverse dialysis for local infusion of compounds. The dialysis membrane is a substantial barrier to diffusion, and concentrations just outside the probe are ≤10% of the perfusate levels with a further exponential decrease as a function of distance from the probe [3]. Moreover, the dialysis probe is a small localized source, and infused compounds must diffuse through a relatively large area of tissue surrounding the probe to produce significant effects on synaptic transmission.To study the role of GABAergic afferents in mediating DAMGO-induced increases in 5-HT efflux, we infused the GABAA receptor antagonist bicuculline into the DRN. Increased extracellular 5-HT during bicuculline infusion suggests that endogenous GABA has a strong tonic inhibitory influence on 5-HT neurons in the rat DRN. This observation is consistent with prior microdialysis studies [28,29]. Increased GABA transmission mediates sleep- related decreases in 5-HT neuronal activity [4,15]. However, GABAA receptor antagonists increased the discharge of 5- HT neurons in the DRN of awake rats, supporting the conclusion that endogenous GABA has a tonic inhibitory influence even during behavioral arousal [4]. Along with 5- HT autoreceptors, GABAergic afferents in the DRN may play a role in negative feedback control of 5-HT neuronal activity [16]. The relatively large increase in 5-HT efflux in response to GABAA receptor antagonists suggests that this is at least as important as somatodendritic autoreceptors in restraining the activity of 5-HT neurons in the DRN under physiological conditions. The present results are consistent with the disinhibition hypothesis of stimulatory effects of A-opioids [8,36]. Thus, pretreatment with bicuculline prevented A-opioid-induced increases in extracellular 5-HT presumably because 5-HT neurons were already disinhibited by pharmacological block- ade of GABAA receptors. In support of the disinhibition hypothesis, GABA-containing neurons in the rat ventral PAG are contacted by enkephalin-containing nerve terminals [35] and express A-opioid receptors [11,13]. Moreover, DAMGO reduced IPSCs recorded in vitro from 5-HT neurons in the DRN [9]. Together, this provides evidence that increased 5- HT efflux in response to DAMGO reflects A-opioid-receptor- mediated inhibition of GABAergic afferents. 4.2. Interactions between opioids, glutamate and 5-HT In agreement with our previous report [28], glutamate receptor antagonists produced small decreases in extracellular 5-HT in the DRN, indicating a weak tonic excitatory influence of endogenous glutamate. Prior blockade of GABAA receptors enhanced this effect, suggesting that the excitatory influence of glutamate is restrained by endoge- nous GABA. This also suggests that increased 5-HT efflux in response to bicuculline resulted partly from disinhibition of excitatory influences on 5-HT neurons. The prefrontal cortex, a region implicated in affective disorders and drug addiction, is one source of glutamatergic afferents to the DRN [14,17]. Other glutamatergic projections are from the lateral habenula, hypothalamus, ventrolateral PAG and medulla [9,10,14]. Glutamatergic inputs mediate phasic sensory stimuli-induced changes in discharge of 5-HT neurons in the cat DRN [15]. Glutamatergic inputs to the DRN also may indirectly regulate 5-HT neurons by stimulation of GABAergic interneurons [2,6,33,34]. Block- ade of this inhibitory pathway might also contribute to the enhanced excitatory influence of endogenous glutamate during infusion of bicuculline. Our data suggest that glutamatergic afferents also are involved in mediating A-opioid effects on 5-HT neurons in the DRN. When GABAA receptors were blocked, DAMGO reduced 5-HT efflux from the level just before infusion of the A-opioid. Glutamate receptor antagonists prevented this reduction. This provides evidence that DAMGO does not directly inhibit 5-HT efflux, a conclusion generally con- sistent with results of recording 5-HT neurons in vitro. Thus, A-opioid receptor stimulation did not directly affect intrinsic membrane properties of most 5-HT neurons recorded in DRN slices [9]. However, enkephalin via A- opioid receptor activation slightly hyperpolarized some 5- HT neurons, suggesting that a direct inhibitory influence cannot be entirely discounted [9]. Non-specific effects of infusing multiple drugs might also account for the DAMGO-induced decrease in 5-HT efflux in the presence of GABAA receptor blockers. Instead, based on the observation that DAMGO greatly reduced evoked EPSCs recorded from 5-HT neurons in DRN slices [9], it seems more likely that a decrease in excitatory input is the major factor in the reduction in 5-HT efflux. In support of this, the present results and our previous report [28] indicate that combined infusion of GABAA and glutamate receptor antagonists into the DRN increases extracellular 5-HT to ¨170% of baseline, the same effect as DAMGO alone. Moreover, during combined infusion of GABAA and glutamate receptor blockers, extracellular 5-HT remained at ¨170% of baseline when DAMGO was added to the aCSF. In summary, the effect of the A-opioid receptor agonist is the same as and non-additive with the effect of blocking GABAA and ionotropic glutamate receptors in the DRN. These results support the hypothesis that A-opioids inhibit both GABAergic and glutamatergic afferents to 5- HT neurons in the DRN. However, in the absence of bicuculline, glutamate receptor antagonists attenuated the increase in 5-HT efflux induced by DAMGO in the DRN. This agrees with our report that glutamate receptor antagonists attenuated the effect of systemic morphine [26]. These observations suggest that stimulation of glutamatergic afferents contributes to the increase in 5- HT efflux elicited by A-opioids. This may reflect dis- inhibition of excitatory influences on 5-HT neurons. According to this hypothesis, DAMGO inhibits GABA- mediated restraint of glutamatergic afferents, thus indi- rectly stimulating 5-HT neurons. In support of this hypothesis, glutamatergic influences on 5-HT in the DRN were weak unless GABAA receptors were blocked. In summary, A-opioid-induced increases in 5-HT efflux represent a balance between competing effects. The inhibition of endogenous GABAergic afferents, which disinhibits 5-HT neurons in part by enhancing the excitatory influence of glutamate, is attenuated by direct inhibition of glutamatergic afferents. The balance between A-opioid influences on glutamatergic and GABAergic afferents favors a small increase in extracellular 5-HT under our experimental conditions. Conceivably, endoge- nous opioids, by inhibiting both inhibitory and excitatory synaptic inputs, block responses of 5-HT neurons to physiological stimuli without causing large changes in baseline discharge rate. Alternatively, in contrast to the widespread effect of infusing an exogenous ligand, release of endogenous opioids in response to specific physiolog- ical stimuli might selectively inhibit either GABAergic or glutamatergic afferents to 5-HT neurons. 4.3. Effect of j-opioids on 5-HT In contrast to A-opioids, U-50,488 decreased extracel- lular 5-HT in the DRN, consistent with our previous report that this n-agonist dose-dependently inhibits 5-HT efflux [27]. We observed near maximal decreases in 5-HT during U-50,488 infusion at a concentration of 300 AM in the aCSF, an effect that was blocked by the selective n-opioid receptor antagonist nor-binaltorphimine [27]. The decrease in extracellular 5-HT was not blocked by GABAA or ionotropic glutamate receptor antagonists. This is consis- tent with evidence that n-opioids in other areas of the CNS directly inhibit principal neurons and thus counteract the disinhibitory effects of A-opioids [18,23]. Infusing U- 50,488 into the NAcc also decreased extracellular 5-HT in this forebrain site [27], consistent with other evidence that n-receptors are present on nerve endings in addition to cell bodies [21]. The molecular mechanisms underlying n-Opioid- induced decreases in 5-HT have not been determined. n- Opioids might inhibit the activity of proteins involved in vesicle exocytosis or by stimulating reuptake as demon- strated for the effect of U-50,488 on dopamine [32]. An increase in the rate of clearance could reduce extracellular 5-HT in the DRN independent of direct n-opioid effects on electrical properties of 5-HT neurons. In turn, decreased activation of somatodendritic autoreceptors might result in increased 5-HT neuronal discharge and release in forebrain projection sites. Alternatively, n-opioids might inhibit 5-HT neuronal discharge. Consistent with this second hypothesis, U-50,488 infusion into the DRN produced decreases in extracellular 5-HT in the NAcc, a forebrain site innervated by 5-HT projections from the DRN. This suggests that changes in extracellular 5-HT in the DRN during infusion of n-opioids reflect 5-HT neuronal discharge and release in forebrain projection sites. Although n-opioids directly inhibit principal neurons in some brain sites [18], a subpopulation of GABA-containing interneurons expresses n-opioid receptors in the medullary raphe nuclei [12]. Thus, although there is no direct anatomical or electrophysiological evidence supporting this possibility, n-opioids might also inhibit GABAergic affer- ents to 5-HT neurons in the DRN. Consistent with this possibility, the reduction in 5-HT in response to n-opioids was enhanced when GABAA receptors were blocked. Assuming that GABAergic interneurons also restrain excitatory influences, this could explain the enhancement of n-opioid-induced decreases in 5-HT during combined infusion of AP-5 and DNQX. Thus, n-mediated inhibition of GABA would lead to increased excitation of 5-HT neurons, and this would partly offset the inhibition of 5-HT release by n-opioids. However, we did not observe a significant enhancement of the effect of U-50,488 during combined infusion of GABAA and glutamate receptor blockers. Moreover, n-opioids depressed EPSPs [20] and did not alter IPSCs recorded from 5-HT neurons in DRN slices [9]. Together, these results suggest that, even if n- opioids influence presynaptic inputs in the DRN, the decrease in 5-HT efflux does not depend on changes in glutamatergic and GABAergic synaptic input.

In conclusion, our results indicate that A- and n-opioids have opposite effects on 5-HT neurons in the rat DRN. The stimulatory influence of A-opioids represents the balance between inhibition of a tonic GABAergic and weaker glutamatergic influence on 5-HT neurons. In contrast, n- opioids reduced 5-HT efflux, and this effect did not depend on changes in GABAergic and glutamatergic afferent input. Endogenous opioids are implicated in adaptive responses to stress such as decreased sensitivity to pain. However, intensely noxious stimuli may induce opioid-dependent changes in afferent regulation of 5-HT neurons in the rat DRN resulting in maladaptive behaviors such as deficits in escape behavior [5]. Thus, understanding the details of the neural circuitry regulating 5-HT neurons might provide new insights into treatment of stress-related behavioral disorders such as depression and drug addiction.