Vol. 290, Issue 3, 1307-1315, September 1999

Modulation of Neurotransmitter Release in the Basal Ganglia of the Rat Brain by Dynorphin Peptides¹

Departments of Physiology and Pharmacology (Z.-B.Y., M.H-M.) and Clinical Neuroscience (Z.-B.Y., L.T.), Karolinska Institutet, Stockholm, Sweden

	Abstract

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Microinjection studies have found that although dynorphin peptides decrease dopamine release in the rat basal ganglia, the nonselective opiate antagonist naloxone produces the opposite effect. To investigate the contribution of the dynorphin pathways to a tonic modulation of dopamine release, a microdialysis study was undertaken, with probes implanted in the substantia nigra and the ipsilateral neostriatum. Perfusion of the substantia nigra with the nonselective antagonist naltrexone (NTX; 1-10 µM), the selective -opoid receptor antagonist, nor-binaltorphimine (nor-BNI; 1-10 µM), and the selective µ-opioid receptor antagonist, D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; 1-10 µM) produced an increase in dopamine release, both in substantia nigra and neostriatum. nor-BNI also produced an increase in dynorphin B release, and a similar effect was observed with the higher concentration of NTX (10 µM). At the higher concentration of NTX and CTOP, an increase in glutamate release was also observed. Perfusion of the neostriatum with NTX, nor-BNI, or CTOP increased striatal dopamine, and dynorphin B release and increased dynorphin B in the ipsilateral substantia nigra. NTX and CTOP, but not nor-BNI, increased striatal glutamate and aspartate release. The -opioid agonist U-50,488H (10 µM) induced a decrease in dopamine levels, both in the substantia nigra and neostriatum, and a paradoxical increase in striatal aspartate levels. Finally, systemic administration of NTX (4 mg/kg s.c.) in awake animals significantly increased striatal dopamine levels. The results suggest that opioid peptides, either dynorphins acting on -opioid receptors or enkephalins acting on µ-opioid receptors, exert tonic inhibition on dopamine and dynorphin B release in both substantia nigra and neostriatum.

	Introduction

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In a previous study (You et al., 1996b), we found that systemic, as well as local, administration of naloxone induced a significant increase in nigral and striatal dopamine (DA) release, suggesting that opioid peptides are tonically active under physiological conditions. However, it is not yet clear which are the receptors involved in this action, because naloxone is a nonselective antagonist (see Martin, 1984). There is general agreement that stimulation of -opioid receptors (Mulder et al., 1984; Herrera-Marschitz et al., 1986: Reid et al., 1988; Di Chiara and Imperato, 1988; Ronken et al., 1993; Schlösser et al., 1995) inhibits DA release, although it has been suggested that µ-opioid receptor activation instead leads to an increase in DA release (Di Chiara and Imperato, 1988). Spanagel et al. (1990, 1992) have reported that both µ- and -opioid receptor agonists increase DA release in the nucleus accumbens. The increase in DA release by -opioid agonists appears to be region-specific, because the -opioid agonists (D-Ala²)deltorphin II (Longoni et al., 1991) and (D-Pen²,D-Pen⁵)enkephalin (Manzanares et al., 1993) increase DA, L-dopa, and/or its metabolites in the nucleus accumbens, but not in the neostriatum. However, it has been recently shown that naltrindole, a selective -opioid receptor antagonist, reduces the increase of DA release elicited by amphetamine in the neostriatum, but not in the nucleus accumbens (Schad et al., 1996).

A prime candidate for the tonic inhibitory effect of opioids on DA release is dynorphin (Dyn) acting on -opioid receptors. The nigrostriatal DA neurons receive reciprocal inputs from striatonigral Dyn-containing neurons. Dyn cell bodies are found throughout the neostriatum of the rat, mainly in the dorsal region, with terminals densely innervating the pars reticulata of the ipsilateral substantia nigra and the entopeduncular nucleus (Vincent et al., 1982; Christensson-Nylander et al., 1986). Axon collaterals within the neostriatum make symmetric contacts with local neurons (Penny et al., 1988). In the substantia nigra, Dyn and -aminobutyric acid (GABA) terminals form synapses with cell bodies and dendrites of DA neurons (Vincent et al., 1982; Van den Pol et al., 1985; Christensson-Nylander et al., 1986). In the neostriatum, Dyn appears to be colocalized with GABA (see Reiner and Anderson, 1990), in neurons which also contain the D₁ DA receptor (Gerfen et al., 1990, 1991) and the cyclic AMP-regulated phosphoprotein DARPP-32 (Langley et al., 1997). It should be noted that dynorphin peptides are partly converted metabolically to Leu-enkephalin-Arg⁶ and Leu-enkephalin in the dopaminergic neurons (Christensson-Nylander et al., 1986; Herrera-Marschitz et al., 1986). A  antagonist may therefore not completely block the signal from these neurons. However, the contribution of enkephalins from the enkephalinergic neurons is much larger and therefore likely more functionally important.

To investigate the role of peptides acting on - or on µ-opioid receptors, we have studied the effect of selective opioid antagonists on DA, Dyn B, Glu, and Asp release in the substantia nigra and neostriatum of the rat with in vivo microdialysis (Ungerstedt et al., 1982). nor-Binaltorphimine (nor-BNI) was chosen as a selective -opioid receptor antagonist (Portoghese et al., 1987), and D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP) as a selective µ-opioid receptor antagonist (Toll, 1992). Naltrexone (NTX) was chosen as a potent but nonselective opioid receptor antagonist (Zukin et al., 1982), and for comparison, U-50,488H, a selective -opioid receptor agonist (Lahti et al., 1982), also was investigated.

	Materials and Methods

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In Vivo Microdialysis. Male Sprague-Dawley rats (ALAB, Stockholm, Sweden) weighing 350 to 450 g were anesthetized with a mixture of air and halothane and placed in a Kopf stereotaxic frame, and two microdialysis probes (CMA/Microdialysis AB, Stockholm, Sweden) were stereotaxically implanted, one into the left striatum (dialyzing length, 4 mm; diameter, 0.5 mm; coordinates: A, 0.5; L, 3.5, V, 8.0, according to Paxinos and Watson, 1982) and the other into the left substantia nigra (dialyzing length, 2 mm; diameter, 0.5 mm; coordinates, A, 6.0; L, 7.5; V, 9.0, inserted with a 40° angle from the vertical in the coronal plane). For experiments in awake animals, striatally implanted microdialysis probes were fixed to the skull with stainless steel screws and methylacrylic cement. The experiments were then performed 24 to 48 h after the implantation, approximately 200 min after the animals were reconnected to the perfusion system.

The microdialysis probes were perfused with a modified cerebrospinal fluid solution (148 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl₂, and 0.85 mM MgCl₂). Two hundred minutes after the implantation of the microdialysis probes, nor-BNI-2HCl (mw 734.78), CTOP (mw 1062), naltrexone-HCl (mw 377.9), or (±)-trans-U-50,488H methanesulfonate (mw 465.44) (Research Biochemicals International, Natick, MA) were perfused through the microdialysis probe, into the substantia nigra, or into the neostriatum, respectively, for a 40-min period.

The drugs were first dissolved in H₂O, and then diluted in the cerebrospinal fluid solution. The perfusion medium was adjusted to pH 7 when required. Changes in the perfusion medium were performed with a syringe selector coupled to a microfraction collector. When administered systemically, NTX was dissolved in saline and injected s.c. in a volume of 1 ml/kg body weight. The rats were maintained under halothane anesthesia throughout the experiment, by free breathing into a mask fitted over the nose (1% halothane in an air flow of 1.5 l/min), unless otherwise indicated. Body temperature was maintained at 37°C by using a temperature-control system. Samples were collected every 40 min (80 µl) and split for analysis of Dyn B (50 µl), catecholamines (10 µl), and amino acids (10 µl).

The experimental protocols were approved by the National Committee for Ethics of Experiment with Laboratory Animals.

Dyn B Radioimmunoassay. The determination of Dyn B was carried out as reported (You et al., 1994a). Briefly, samples (50 µl of perfusate) and standards diluted in the perfusion medium were incubated with the antiserum and the labeled peptide in Eppendorf polyethylene tubes for 24 h at 4°C. Samples without antiserum (to determine nonspecific binding) and samples without unlabeled peptide (to determine maximal tracer binding) were simultaneously incubated. Dyn B (Bachem, Bubendorf, Switzerland) was labeled with ¹²⁵I using a chloramine-T procedure and purified by reversed-phase HPLC with a gradient of 15 to 40% acetonitrile containing 0.04% trifluoroacetic acid. The antiserum and the labeled peptide used in the assay were dissolved in 0.05 M phosphate buffer containing 0.15 M NaCl, 0.1% gelatin, 0.1% BSA, 0.02% sodium azide, and 0.1% Triton X-100. After incubation, the antibody-bound and free tracer were separated by addition of anti-rabbit IgG coupled to Sepharose (Pharmacia Decanting Suspension 3; Pharmacia AB, Uppsala, Sweden) and centrifugation for 15 min in a Beckman Microfuge. The bound fraction was counted in a gamma counter. The detection limit was 0.1 to 0.2 fmol/tube (2 pM). The nonspecific binding of the tracer in the absence of the antiserum was less than 2%.

Determination of Catecholamines. DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and the serotonin metabolite 5-hydroxy-indoleacetic acid (5-HIAA) were measured with HPLC coupled to electrochemical detection. The detection limit was 0.2 nM for DA, DOPAC, and 5-HIAA and 1 nM for HVA (see Herrera-Marschitz et al., 1996).

Determination of Amino Acids. Glu and Asp were measured with HPLC with precolumn derivatization with a o-phthaldialdehyde/mercaptoethanol reagent and fluorescence detection. The detection limit for both Glu and Asp was 10 nM (see Herrera-Marschitz et al., 1992, 1996).

In Vitro Recovery. The 2- and 4-mm microdialysis probes used in this study showed 14 and 20% in vitro recovery for DA, DOPAC, HVA, 5-HIAA, Glu, and Asp, and 6 and 9%, for Dyn B, respectively.

Histology. After completion of the microdialysis experiments, the rats were sacrificed with an overdose of halothane, and the brain was dissected out. The probe location was examined at low magnification with a surgical microscope. Only animals with correctly implanted probes are included in the statistics.

Statistics. The levels of the assayed substances are expressed as the concentrations found in the perfusates (means ± S.E.). Basal values refer to the values obtained before the inclusion of the drugs into the perfusion medium and are set as 100%. The dose dependence of the effects was analyzed with Fisher-ANOVA. Drug effects in the respective groups were tested with the paired Student's t test. A level of P < .05 for a two-tailed test was considered critical for statistical significance.

	Results

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NTX. Nigral perfusion with NTX (1-10 µM; Table 1) increased extracellular levels of DA in the substantia nigra and neostriatum. After the higher concentration of NTX (10 µM), DOPAC, HVA, Glu, and Dyn B levels were also increased. The increase in DOPAC and HVA levels was less pronounced than that of DA. Figure 1 shows the time course of the effect of 10 µM NTX on nigral (a) and striatal (b) DA and Dyn B levels. The effect on striatal DA release was strong and long-lasting.

View this table: [in this window] [in a new window]   TABLE 1 Effect of nigral of naltrexone perfusion on extracellular Dyn B, DA, DOPAC, HVA, glutamate, and aspartate levels (mean ± S.E.) measured with microdialysis in halothane-anesthetized rats The maximum effect observed among three successive samples taken immediately after drug administration is expressed as the percentage of the respective basal value; N = 6 for both conditions.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   a and b, effect of nigral perfusion for 40 min with 10 µM NTX on extracellular dopamine (circles) and dynorphin B (squares) levels in the substantia nigra (a) and neostriatum (b). Triangles indicate dopamine or dynorphin B (inverted triangles) levels measured without any drug added to the perfusion medium. Levels are expressed as the percentage of the respective basal values (N = 5-6 for each group; dotted line = 100%; *P <.05).

Striatal perfusion with NTX (1-10 µM) (Table 2) induced a prominent increase in striatal DA levels without affecting those in the ipsilateral substantia nigra. The effect on DA levels was dependent on the concentration of NTX (Table 2). Dyn B levels were also increased, both in neostriatum and substantia nigra, whereas Glu and Asp levels were only significantly increased in the neostriatum. The time course of the effect of 10 µM NTX on striatal (Fig. 2a) and nigral (Fig. 2b) DA and Dyn B release showed a strong effect on striatal DA release.

View this table: [in this window] [in a new window]   TABLE 2 Effect of striatal naltrexone perfusion See legend to Table 1 for experimental details. N = 4-5 (1 µM) and 5 (10 µM).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   a and b, effect of striatal perfusion for 40 min with 10 µM NTX on extracellular dopamine (circles) and Dyn B (squares) levels in the neostriatum (a) and substantia nigra (b). (See also legend to Fig. 1; N = 5-6 for each group).

Nor-BNI. Nigral perfusion with nor-BNI (1-10 µM; Table 3) produced an increase in DA and Dyn B levels in both the substantia nigra and neostriatum. The effect on Dyn B levels was long-lasting. Figure 3 shows the time course of the effect of 10 µM nor-BNI infused for 40 min on nigral (a) and striatal (b) DA and Dyn B levels.

View this table: [in this window] [in a new window]   TABLE 3 Effect of nigral nor-BNI perfusion See Table 1 legend for experimental details. N = 5 for both conditions.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   a and b, effect of nigral perfusion for 40 min with 10 µM nor-BNI on extracellular dopamine (circles) and Dyn B (squares) levels in the substantia nigra (a) and neostriatum (b). (See also legend to Fig. 1; N = 5-6 for each group).

Striatal perfusion with nor-BNI (1-10 µM; Table 4) produced a local increase in DA levels. A prominent effect on Dyn B levels was observed both in neostriatum and substantia nigra. The time course of the effect of 10 µM nor-BNI on DA and Dyn B release in the neostriatum and substantia nigra is shown in Fig. 4, a and b.

View this table: [in this window] [in a new window]   TABLE 4 Effect of striatal nor-BNI perfusion See Table 1 legend for experimental details. N = 5 for all experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   a and b, effect of striatal perfusion for 40 min with 10 µM nor-BNI on extracellular dopamine (circles) and Dyn B (squares) levels in the neostriatum (a) and substantia nigra (b). (See also legend to Fig. 1; N = 5-6 for each group).

CTOP. Nigral perfusion with CTOP (1-10 µM; Table 5) produced a local increase in DA levels. At the higher concentration of CTOP, an effect on striatal DA and Glu levels was also observed.

View this table: [in this window] [in a new window]   TABLE 5 Effect of nigral CTOP perfusion See Table 1 legend for experimental details. N = 5 for all experiments.

Striatal CTOP (1-10 µM; Table 6) increased DA, Glu, and Asp levels locally. Dyn B levels were increased both in the neostriatum and substantia nigra, but only after administration of 1 µM of CTOP (Table 6).

View this table: [in this window] [in a new window]   TABLE 6 Effect of striatal CTOP perfusion See Table 1 legend for experimental details. N = 5 (1 µM) or 6 (10 µM).

U-50,488H. Nigral perfusion with the  agonist U-50,488H (10 µM; Table 7) produced a 20% decrease in local DA and DOPAC levels. When administered into the neostriatum, U-50,488H (10 µM) produced a similar decrease in DA levels (20%) and a prominent increase (>200%) in striatal Asp without affecting Glu levels.

View this table: [in this window] [in a new window]   TABLE 7 Effect of nigral or striatal U-50,488H (10 µM) perfusion See Table 1 legend for experimental details.

Systemic Naltrexone Treatment. NTX administered s.c. at the dose of 4 mg/kg under awake conditions produced a significant increase in striatal DA levels (Table 8). Dyn B levels also were increased, but the statistic criterion was not reached.

View this table: [in this window] [in a new window]   TABLE 8 Effect of systemic NTX (4 mg/kg s.c.) on extracellular Dyn B, DA, DOPAC, HVA, GABA, glutamate, and aspartate levels (mean ± S.E.) in awake animals The drug was administered 24 to 48 h after the implantation of the microdialysis probe, approximately 200 min after the reconnection to the perfusion system. The maximum effect observed among three successive samples taken immediately after the drug administration is expressed as the percentage of the respective basal value. N = 3-7.

	Discussion

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Modulation of Dopamine Release by Endogenous Opioid Peptides. All intracerebrally administered opioid antagonists induced a local increase in extracellular DA levels. Striatal DA levels were also increased by systemic administration of NTX under awake conditions. A similar effect on DA transmission has been observed after local or systemic administration of naloxone (You et al., 1996a). The effect of NTX on DA was, however, more pronounced, which is to be expected, because NTX has a greater potency and longer duration than naloxone (Zukin et al., 1982). These observations confirm the hypothesis that blockade of opioid receptors results in a disinhibition of the nigrostriatal DA pathway (You et al., 1996a), as also proposed by several authors using in vitro and in vivo approaches (see Feigenbaum and Howard, 1996). There are, however, studies reporting that blockade of opioid receptors with naloxone inhibits amphetamine-induced DA release (Schad et al., 1995, 1996; Kimmel et al., 1998), but this effect appears to be restricted to stimulated DA release (see Pothos et al., 1991), produced by rather high concentrations of naloxone, and mainly affecting DA release in nucleus accumbens. In agreement, it has been reported that µ- as well as -opioid receptor agonists increase DA release in nucleus accumbens, but not in neostriatum (Spanagel et al., 1990, 1992; Longini et al., 1991; Manzanares et al., 1993).

In the substantia nigra, -opioid receptor mRNA is mainly expressed by cells in the pars compacta (see Mansour et al., 1994a, 1995), but  receptor-binding sites are also abundant in the pars reticulata, probably on DA dendrites. Local injection of Dyn peptides into the pars reticulata of the substantia nigra produces a concentration-dependent decrease in DA release (Herrera-Marschitz et al., 1986; Reid et al., 1988; Di Chiara and Imperato, 1988) and, as shown here, this effect is also observed after local administration of the  agonist U-50,488H (Lahti et al., 1982). On the contrary, the selective -opioid antagonist nor-BNI (Portoghese et al., 1987) produced an increase in DA release. CTOP, a selective µ-opioid receptor antagonist (Toll, 1992), also produced an increase in DA release, although the effect was less strong than that produced by nor-BNI. µ-Opioid receptors have been observed in both pars compacta and pars reticulata of the substantia nigra (Sharif and Hughes, 1989; Mansour et al., 1994b), probably on DA neurons, because the number of µ-opioid receptor-binding sites is decreased after a lesion of the nigro-striatal DA pathway (Bodnar et al., 1988).

In the neostriatum, -opioid receptors have been localized presynaptically on DA terminals, both by receptor autoradiography (Sharif and Hughes, 1989), and by functional studies (Mulder et al., 1984; Werling et al., 1988; Jackisch et al., 1993). Thus, the effect of striatal nor-BNI on DA release is probably due to a direct disinhibition of -opioid receptors located on DA terminals. µ-Opioid receptor mRNA and binding sites have been observed in the patch compartment of the neostriatum (see Mansour et al., 1995). These receptors may also be located on DA terminals, because the density of µ-opioid receptor-binding sites is decreased in the neostriatum of rats with a unilateral 6-hydroxydopamine lesion (Smith et al., 1993) and is reduced in the caudate nucleus of patients suffering from Parkinson's disease (Fernandez et al., 1994). This decrease in µ-opioid receptor may also indicate a trans-synaptic regulation of µ-opioid receptor gene expression by DA release (Smith et al., 1993).

CTOP administered into the neostriatum produced an increase in striatal DA levels, further suggesting an effect mediated by µ-opioid receptors directly on DA neurons, or indirectly on corticostriatal neurons, which provide an excitatory input, as proposed by Jiang and North (1995; see also Wong et al., 1996). In fact, it was found here that local perfusion with NTX or CTOP significantly increased Glu levels. An increase in excitatory amino acid levels may then contribute, at least in part, to the increase in DA release, which can be induced by electrical and chemical stimulation of the neocortex (Nieoullon et al., 1978; Herrera-Marschitz, 1992; Taber and Fibiger, 1993), local inhibition of Glu re-uptake (Herrera-Marschitz et al., 1992), and perfusion with L-Glu (Chéramy et al., 1986).

Modulation of Dyn B Release. Dyn-containing cell bodies are found throughout the neostriatum, mainly in the dorsal region, with terminals densely innervating the pars reticulata of the ipsilateral substantia nigra and the entopeduncular nucleus (Vincent et al., 1982; Christensson-Nylander et al., 1986), and with axon collaterals making symmetric contacts with local neurons in the neostriatum, (Penny et al., 1988). Dyn appears to be colocalized with GABA in these neurons (see Reiner and Anderson, 1990). It has been shown that glutamic acid decarboxylase (GAD)-immune reactivity (IR) and Dyn-IR terminals make synaptic contacts with (Van den Pol et al., 1985), or surround tyrosine hydroxylase (TH)-IR cell bodies and dendrites of the substantia nigra (Christensson-Nylander et al., 1986). Dyn peptides are released in a Ca²⁺-dependent manner in both substantia nigra and neostriatum and may act to further modulate the inputs and outputs of the basal ganglia (You et al., 1994a).

In the substantia nigra, nor-BNI and NTX induced a significant increase in extracellular Dyn B levels. The effect of nor-BNI was generally more pronounced than that of NTX. In a previous study, we found that DA, via D₁ receptors, exerts a stimulatory effect on Dyn B release in substantia nigra and in neostriatum (You et al., 1994b). However, it is unlikely that the increase in nigral Dyn B levels produced by nor-BNI or NTX is secondary to an increase in DA release, because CTOP administered into the substantia nigra increased DA release, but had no effect on Dyn B.

In the neostriatum, all opioid antagonists produced an increase in Dyn B and DA release. The concentration-dependent effects of nor-BNI and NTX were followed by an increase of DynB levels in the ipsilateral substantia nigra, suggesting a direct effect on Dyn-containing striatonigral neurons.

The  receptor agonist U-50,488H did not affect Dyn B, whereas it decreased DA release and increased Asp levels in the neostriatum. It may be argued that the concentration used for U-50,488H was not high enough for decreasing, as expected, Dyn B release. Indeed, relative to previous studies (Reid et al., 1988; Herrera-Marschitz et al., 1989, 1990), the effect of U-50,488H on DA release was minor. It may also happen that the strong effect on Asp levels antagonized the effect of U-50,488H, because we have previously observed that striatal elevation of Asp levels is followed by an increase in Dyn B release both in neostriatum and in ipsilateral substantia nigra (You et al., 1996b).

Modulation of Extracellular Glutamate and Aspartate Levels. NTX and CTOP produced an increase in extracellular Glu and/or Asp levels in the substantia nigra and neostriatum, suggesting a tonic inhibition via µ-receptors. These effects were never observed after nor-BNI, ruling out an action on  receptors. Thus, enkephalin, either derived from proenkephalin or generated indirectly from enzymatic processing of Dyn peptides, seems to exert a tonic inhibitory modulation on Glu- and Asp-containing terminals.

As indicated above, striatal administration of U-50,488H produced a strong increase in striatal Asp levels without any significant effect on Glu. Activation of Asp release independent of Glu has been previously observed and related to an intrinsic Asp neuronal system in the neostriatum (You et al., 1994c, 1996b; Pettersson et al., 1996). Dyn collaterals in the neostriatum may exert a stimulatory modulation of this intrinsic Asp system, via -receptors, probably involving a polysynaptic disinhibitory loop.

	Conclusions

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Tonic inhibitory opioid control of DA release in the rat basal ganglia involves both  and µ receptors. The release of Dyn peptides is also subjected to tonic modulation by these receptors. This is in contrast to the findings of Spanagel et al. (1992), who observed tonic stimulation of DA release via µ-receptors in the VTA, which was ascribed to disinhibition of a GABA-containing interneuron and tonic inhibition via  receptors in the nucleus accumbens, cell body, and terminal region, respectively, for mesolimbic DA neurons. The release of excitatory amino acids in the neostriatum appears to be under tonic inhibitory modulation via µ receptors, whereas Asp is released via  receptors, probably via disinhibitory mechanisms. The modulation via -opioid receptor subtypes remains to be elucidated.

In conclusion, the modulatory activity of opioid peptides in the basal ganglia is mainly inhibitory, with one notable exception being the release of Asp, probably from an intrinsic neuronal system.

	Footnotes

Accepted for publication May 2, 1999.

Received for publication January 13, 1999.

¹ This study was supported by grants from the Swedish Medical Research Council (8669, 10797, 3766, and 3096), the National Institute on Drug Abuse (Rockville, MD), and the Karolinska Institutet Fonder, the Swedish Society of Medicine and Swedish Match. Z.-B.Y. was a recipient of a Karolinska Institutet fellowship.

Send reprint requests to: Dr. Mario Herrera-Marschitz, Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden. E-mail: mario.herrera-marschitz{at}fyfa.ki.se

	Abbreviations

DA, dopamine; CTOP, D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂; Dyn, dynorphin; GABA, -aminobutyric acid; GAD, glutamic acid decarboxylase; IR, immunoreactivity; nor-BNI, nor-binaltorphimine; NTX, naltrexone; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid.

	References

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

• Bodnar RJ, Clark JA, Cooper ML and Pasternal GW (1988) Loss of striatal Mu1 opiate receptor binding by substantia nigra lesions in the rat. Life Sci  43: 1697-1700[Medline].
• Chéramy A, Romo R, Godeheu G, Baruch P and Glowinski J (1986) In vivo presynaptic control of dopamine release in the cat caudate nucleus-II. Facilitatory or inhibitory influence of L-glutamate. Neuroscience  19: 1081-1090[Medline].
• Christensson-Nylander I, Herrera-Marschitz M, Staines W, Hökfelt T, Terenius L, Ungerstedt U, Cuello C, Oertel WH and Goldstein M (1986) Striato-nigral dynorphin and substance P pathways in the rat. I. Biochemical and immunohistochemical evidence. Exp Brain Res  64: 169-192[Medline].
• Di Chiara G and Imperato A (1988) Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats. J Pharmacol Exp Ther  244: 1067-1080[Abstract/Free Full Text].
• Feigenbaum JJ and Howard SG (1996) The effect of naloxone on spontaneous and evoked dopamine release in the central and peripheral nervous system. Life Sci  24: 2009-2019.
• Fernandez A, de Ceballos ML, Jenner P and Marsden CD (1994) Neurotensin, substance P, delta and mu opioid receptors are decreased in basal ganglia of Parkinson's disease patients. Neuroscience  61: 73-79[Medline].
• Gerfen CR, Engber TM, Mahan LC, Suzel Z, Chase TM, Monsma F and Sibley DR (1990) D1 and D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons. Science (Wash DC)  250: 1429-1432[Abstract/Free Full Text].
• Gerfen CR, McGinty JF and Oung WS (1991) Dopamine differentially regulates dynorphin, substance P and enkephalin expression in striatal neurons: In situ hybridization histochemical analysis. J Neurosci  11: 1016-1031[Abstract].
• Herrera-Marschitz M, Christensson-Nylander I, Sharp T, Staines W, Reid M, Hökfelt T, Terenius L and Ungerstedt U (1986) Striato-nigral dynorphin and substance P pathways in the rat. II. Functional analysis. Exp Brain Res  64: 193-207[Medline].
• Herrera-Marschitz M, Meana JJ, O'Connor WT, Goiny M, Reid MS and Ungerstedt U (1992) Neuronal dependence of extracellular dopamine, acetylcholine, glutamate, aspartate and gamma-aminobutyric acid (GABA) measured simultaneously from rat neostriatum using in vivo microdialysis: Reciprocal interactions. Amino Acids  2: 157-179.
• Herrera-Marschitz M, Reid MS and Terenius L (1990) -Casomorphins and the basal gangliaEffects of intranigral administration in rats, in -Casomorphins and Related Peptides (Nyberg F and Brantl V eds) pp 111-115, Fyris-Tryck AB, Uppsala, Sweden.
• Herrera-Marschitz M, Terenius L, Grehn L and Ungerstedt U (1989) Rotational behaviour produced by intranigral injections of bovine and human -casomorphins in rats. Psychopharmacology  99: 357-361[Medline].
• Herrera-Marschitz M, You Z-B, Goiny M, Meana JJ, Silveira R, Godukhin O, Chen Y, Espinoza S, Pettersson E, Loidl F, Lubec G, Andersson K, Nylander I, Terenius L and Ungerstedt U (1996) On the origin of extracellular glutamate levels monitored in the basal ganglia by in vivo microdialysis. J Neurochem  66: 1726-1735[Medline].
• Jackisch R, Holz H and Hertting G (1993) No evidence for presynaptic opioid receptors on cholinergic, but presence of kappa-receptors on dopaminergic neurons in the rabbit caudate nucleus: Involvement of endogenous opioids. Naunyn-Schmiedeberg's Arch Pharmacol  348: 234-241[Medline].
• Jiang ZG and North RA (1995) Pre- and postsynaptic inhibition by opioids in rat striatum. J Neurosci  12: 356-361[Abstract].
• Kimmel HL, Schad CA, Justice JB, Jr and Holtzman SG (1998) Naloxone does not alter amphetamine-induced rotational behaviour or striatal dopamine levels of nigrally-lesioned rats. Brain Res  789: 171-174[Medline].
• Lahti RA, VonVoigtlander PF and Barsuhn C (1982) Properties of a selective  agonist, U-50,488H. Life Sci  31: 2257-2260[Medline].
• Langley KC, Berson C, Greengard P and Quimet CC (1997) Co-localization of the D1 dopamine receptor in a subset of DARPP-32-containing neurons in rat caudate-putamen. Neuroscience  78: 977-983[Medline].
• Longoni R, Spina L, Mulas A, Carboni E, Garau L, Melchiorri P and Di Chiara G (1991) (D-Ala²)Deltorphin II: D₁-dependent stereotypies and stimulation of dopamine release in the nucleus accumbens. J Neurosci  11: 1565-1576[Abstract].
• Mansour A, Fox CA, Akil H and Watson SJ (1995) Opioid-receptor mRNA expression in the rat CNS: Anatomical and functional implications. Trends Neurosci  18: 22-29[Medline].
• Mansour A, Fox CA, Meng F, Akil H and Watson SJ (1994a) ₁ receptor mRNA distribution in the rat CNS: Comparison to  receptor binding and prodynorphin mRNA. Mol Cell Neurosci  5: 124-144[Medline].
• Mansour A, Fox CA, Thompson RC, Akil H and Watson SJ (1994b) µ-opioid receptor mRNA expression in the rat CNS: Comparison to µ-receptor binding. Brain Res  643: 245-265[Medline].
• Manzanares J, Durham RA, Lookingland KJ and Moore KE (1993) -opioid receptor-mediated regulation of central dopaminergic neurons in the rat. Eur J Pharmacol  249: 107-112[Medline].
• Martin WR (1984) Pharmacology of opioids. Pharmacol Rev  35: 283-323[Abstract].
• Mulder AH, Wardeh G, Hogenboom F and Frankhuyzen AL (1984) - and -opioid receptor agonists differentially inhibit striatal dopamine and acetylcholine release. Nature (Lond)  308: 278-280[Medline].
• Nieoullon A, Chéramy A and Glowinski J (1978) Release of dopamine evoked by electrical stimulation of the motor and visual areas of the cerebral cortex in both caudate nuclei and in the substantia nigra in the cat. Brain Res  145: 69-83[Medline].
• Paxinos G and Watson C (1982) The Rat Brain in Stereotaxic Coordinates Academic Press, New York.
• Penny GR, Wilson CJ and Kitai ST (1988) Relationship of the axonal and dendritic geometry of spiny projection neurons to the compartmental organization of the neostriatum. J Comp Neurol  269: 275-289[Medline].
• Pettersson E, Herrera-Marschitz M, Rodriguez-Puertas R, Xu Z-Q, You Z-B, Hughes J, Elde RP, Ungerstedt U and Hökfelt T (1996) Evidence for aspartate-immunoreactive neurons in the neostriatum of the rat: Modulation by the mesencephalic dopamine pathway via the D₁-subtype of receptor. Neuroscience  74: 51-66[Medline].
• Porthogese PS, Lipkowski AW and Takemori AE (1987) Binaltorphimine and nor-binaltorphimine, potent and selective  opioid receptor antagonists. Life Sci  40: 1287-1292[Medline].
• Pothos E, Rada P, Mark GP and Hoebel BG (1991) Dopamine microdialysis in the nucleus accumbens during acute and chronic morphine, naloxone-precipitated withdrawal and clonidine treatment. Brain Res  566: 348-350[Medline].
• Reid M, Herrera-Marschitz M, Hökfelt T, Terenius L and Ungerstedt U (1988) Differential modulation of striatal dopamine release by intranigral injection of -aminobutyric acid (GABA), dynorphin A and substance P. Eur J Pharmacol  147: 411-420[Medline].
• Reid MS, O'Connor TW, Herrera-Marschitz M and Ungerstedt U (1990) The effects of intranigral GABA and dynorphin A injections on striatal dopamine and GABA release: Evidence that dopamine provides inhibitory regulation of striatal GABA neurons via D₂ receptors. Brain Res  519: 255-260[Medline].
• Reiner A and Anderson KD (1990) The patterns of neurotransmitter and neuropeptide co-occurrence among striatal projection neurons: Conclusions based on recent findings. Brain Res Rev  15: 251-265[Medline].
• Ronken E, Mulder AH and Schoffelneer AM (1993) Interacting presynaptic kappa-opioid and GABA_A receptors modulate dopamine release from rat striatal synaptosomes. J Neurochem  61: 1634-1639[Medline].
• Schad CA, Justice JB, Jr and Holtzman SG (1995) Naloxone reduces the neurochemical and the behavioral effects of amphetamine but not those of cocaine. Eur J Pharmacol  275: 9-16[Medline].
• Schad CA, Justice JB, Jr and Holtzman SG (1996) Differential effects of - and µ-opioid receptor antagonists on the amphetamine-induced increase in extracellular dopamine in striatum and nucleus accumbens. J Neurochem  67: 9-16.
• Schlösser B, Kudernatsch MB, Sutor B and ten Bruggencate G (1995) , µ and  opioid receptor agonists inhibit dopamine overflow in rat striatal slices. Neurosci Lett  191: 126-130[Medline].
• Sharif NA and Hughes J (1989) Discrete mapping of brain mu and delta opioid receptors using selective peptides: Quantitative autoradiography, species differences and comparison with kappa receptors. Peptides  10: 499-522[Medline].
• Smith JAM, Leslie FM, Brodie RS and Loughlin SE (1993) Long-term changes in striatal opioid systems after 6-hydroxydopamine lesion of rat substantia nigra. Neuroscience  55: 935-951[Medline].
• Spanagel R, Herz A and Shippenberg TS (1990) The effects of opioid peptides on dopamine release in the nucleus accumbens: An in vivo microdialysis study. J Neurochem  55: 1734-1740[Medline].
• Spanagel R, Herz A and Shippenberg TS (1992) Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway. Proc Natl Acad Sci USA  89: 2046-2050[Abstract/Free Full Text].
• Taber MT and Fibirger HC (1993) Electrical stimulation of the medial prefrontal cortex increases dopamine release in the striatum. Neuropsychopharmacology  9: 356-361.
• Toll L (1992) Comparison of mu opioid receptor binding on intact neuroblastoma cells with guinea pig brain and neuroblastoma cell membranes. J Pharmacol Exp Ther  260: 9-15[Abstract/Free Full Text].
• Ungerstedt U, Herrera-Marschitz M, Jungnelius U, Ståhle L, Tossman U and Zetterström T (1982) Dopamine synaptic mechanisms reflected in studies combining behavioural recordings and brain dialysis. Adv Biosci  37: 219-231.
• Van den Pol AM, Smith AD and Powell JF (1985) GABA axons in synaptic contact with dopamine neurons in the substantia nigra: Double immunocytochemistry with biotin-peroxidase and protein A-colloid gold. Brain Res  348: 146-154[Medline].
• Vincent SR, Hökfelt T, Christensson I and Terenius L (1982) Immunohistochemical evidence for a dynorphin immunoreactive striato-nigral pathway. Eur J Pharmacol  85: 251-252[Medline].
• Werling LL, Frattali A, Portoghese PS, Takemori AL and Cox BM (1988) Kappa receptor regulation of dopamine release from striatum and cortex of rats and guinea pigs. J Pharmacol Exp Ther  246: 282-286[Abstract/Free Full Text].
• Wong H, Moriwaki A, Wang JB, Uhl GR and Pickel VM (1996) Ultrastructural immunocytochemical localization of µ opioid receptors and Leu⁵-enkephalin in the patch compartment of the rat caudate-putamen nucleus. J Comp Neurol  375: 659-674[Medline].
• You Z-B, Herrera-Marschitz M, Nylander I, Goiny M, Kehr J, Ungerstedt U and Terenius L (1996a) Effect of morphine on dynorphin B and GABA release in the basal ganglia of rats. Brain Res  710: 241-248[Medline].
• You Z-B, Herrera-Marschitz M, Nylander I, Goiny M, O'Connor WT, Ungerstedt U and Terenius L (1994b) The striatonigral dynorphin pathway of the rat studied with in vivo microdialysis. II. Effects of dopamine D₁ and D₂ receptor agonists. Neuroscience  63: 427-434[Medline].
• You Z-B, Herrera-Marschitz M, Pettersson E, Nylander I, Goiny M, Shou H-Z, Kehr J, Godukhin O, Hökfelt T, Terenius T and Ungerstedt U (1996b) Modulation of neurotransmitter release by cholecystokinin in the neostriatum and substantia nigra of the rat: Regional and receptor specificity. Neuroscience  74: 793-804[Medline].
• You Z-B, Nylander I, Herrera-Marschitz M, O'Connor WT, Goiny M and Terenius L (1994a) The striatonigral dynorphin pathway of the rat studied with in vivo microdialysis. I. Effects of K⁺-depolarization, lesions and peptidase inhibition. Neuroscience  63: 415-425[Medline].
• You Z-B, Pettersson E, Herrera-Marschitz M, Hökfelt T, Terenius T, Nylander I, Goiny M, Hughes J, O'Connor WT and Ungerstedt U (1994c) Modulation of striatal aspartate and dynorphin B release by cholecystokinin (CCK-8) studied in vivo with microdialysis. Neuroreport  5: 2301-2304[Medline].
• Zukin RS, Sugarman JR, Fitz-Syage ML, Gardner EL, Zukin SR and Gintzler AR (1982) Naltrexone-induced opiate receptor supersensitivity. Brain Res  245: 285-292[Medline].