Buprenorphine Differentially Alters Opioid Receptor Adaptation in Rat Brain Regions

Abstract

Previous In vivo studies revealed that the mixed agonist-antagonist buprenorphine can down-regulate μ and up-regulate δ₂ and κ₁, opioid receptors in rat brain. In this report brain regional differences in opioid receptor adaptation were addressed. Rats received i.p. injections with buprenorphine (0.5–2.5 mg/kg) and were killed 20 h later. Membranes from 7 brain regions were analyzed for μ (³H-[D-Ala²,N-mephe⁴,Gly-ol⁵] enkephalin), κ₁, (³H-U-69593), δ₁(³H-[D-Pen²,D-Pen⁵] enkephalin) and δ₂ (³H-deltorphin II) receptor binding parameters. Buprenorphine induced down-regulation of μ receptors in frontal cortex, occipital cortex, thalamus, hippocampus, striatum and brain stem. K_d values for ³H-[D-Ala²,N-mephe⁴,Gly-ol⁵] enkephalin were unchanged from controls. Up-regulation of κ₁, receptors was observed in frontal, parietal, occipital cortexes and striatum. Binding to δ₂ sites was elevated in frontal and parietal cortexes. Buprenorphine did not alter δ₁ binding in any of the regions examined. Changes in opioid receptor adaptation induced by buprenorphine were further supported by data from cross-linking of ¹²⁵l-β-endorphin to cortical membrane preparations. A reduction in a 60- to 65-kDa band was detected in frontal and occipital cortices in which binding assays revealed down-regulation of μ receptors. In parietal cortex neither the 60- to 65-kDa product nor B_max changes were observed. These results indicate that buprenorphine is a useful tool to study brain opioid receptor adaptation in vivo and the information accrued may be relevant to the mode of action of this drug in the treatment of heroin and cocaine abuse.

Antagonist- and agonist-induced changes in opioid receptor levels have been more readily demonstrated in cell cultures and in developing rat brain than in adult brain (Chang et al., 1982; Bhargava and Gulati, 1990; Tempel et al., 1990; Zadina and Kastin, 1986; Belcheva et al., 1992; Belcheva et al., 1993). Agonist-mediated brain opioid receptor down-regulation in vivo has been more difficult to substantiate. Earlier in vivo down-regulation studies were performed with whole brain membranes. Therefore, opioid binding changes in discrete brain regions, possibly involving specific receptor types or subtypes, may have gone undetected.

Recently, the importance of brain regional specificity for opioid receptor adaptation was discovered. Opioid receptor up- and down-regulation appears to occur only in discrete brain regions and for certain receptor types and subtypes (Belcheva et al., 1993; Belcheva et al., 1994; Dingledine et al., 1983; Loh et al., 1988; Steece et al., 1989; Werling et al., 1989). Receptor autoradiography studies revealed 2-fold increases in opioid binding in several subregions of brain with no change in adjacent subregions after chronic antagonist administration to rats (Tempel et al., 1984; Paden et al., 1987). Similarly, chronic agonist exposure resulted in down-regulation of μ and δ receptors in selective rat brain regions (Bhargava and Gulati, 1990; Tao et al., 1988). The opioid antagonist, naltrexone, elicited a transient down-regulation of δ₂ receptors preceding up-regulation in both brain and NG108-15 cells (Belcheva et al., 1992; Belcheva et al., 1994). Treatment of rats with naltrexone resulted in lowered ³H-DSLET B_max values in hindbrain, but not in striatum, hippocampus or cortex and subsequently 2-fold up-regulation of δ opioid binding in all four regions (Belcheva et al., 1994).

Cross-linking of opioid receptors with ¹²⁵I-β-endorphin has proven useful for the estimation of their molecular sizes (Howard et al., 1985; Keren et al., 1988; McLean et al., 1989; Schoffelmeer et al., 1990; Ko et al., 1992). In β-endorphin competition binding experiments with unlabeled μ- and δ-selective ligands or by using cell lines known to express only certain classes of opioid receptor, it has been possible to associate a 60- to 65-kDa band with μ and a 50- to 55-kDa band with δ sites. NG108-15 cell membranes, which appear to contain only δ receptors, undergo ¹²⁵I-β-endorphin cross-linking to generate 50- to 55-, 38- to 43- and 25- to 27-kDa bands. These and other results suggest the occurrence of limited proteolysis of both μ, and δ opioid receptors. Ko et al. 1992 proposed that only the 25-kDa band fulfilled the requirements for a δ opioid binding site, in part because its intensity alone was reduced under opioid receptor down-regulation conditions in NG108-15 cells. Inasmuch as this 25-kDa band is the predominant species cross-linked in NG108-15 cells but undetectable in brain (Howard et al., 1985; McLean et al., 1989), it was of interest to determine the size of the cross-linked product(s) down-regulated in brain.

When the mixed agonist-antagonist buprenorphine was administered to both adult and neonatal rats, it induced down-regulation of μ and up-regulation of κ₁ and δ₂ brain receptors (Belcheva et al., 1992). We report that buprenorphine administration to rats induces a brain-region-specific down- and up-regulation of the opioid sites. These conclusions are based on homologous competition studies with μ-, δ₁-, δ₂- and κ₁-selective ligands and on ¹²⁵I-β-endorphin cross-linking experiments performed with membrane preparations from rat brain regions.

Materials and Methods

Chemicals

DAMGE, DPDPE and β-endorphin were obtained from Chiron (San Diego, CA), whereas buprenorphine and U69593 were from NIDA Drug Supply (Research Triangle, NC) and deltorphin II was from BACHEM Bioscience Inc. (King of Prussia, PA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or Pierce (Rockford, IL).

Buprenorphine administration to rats

Adult male Sprague-Dawley rats received i.p. injections with either buprenorphine (0.5–2.5 mg/kg) or saline, and 20 hr later the animals were killed. In preliminary experiments a 6-day regimen of daily buprenorphine injections gave the same results for rat forebrain membranes (³H-DAMGE B_max =54.7 ± 4.2 and 19.9 ± 1.1 fmol/mg prot. P < .05 for controls vs. 2.5 mg/kg buprenorphine-treated, respectively) as the single 20-hr treatment (³H-DAMGE B_mM =70.1 ± 5.6 and 16 ± 2.4 fmol/mg prot. P < .05, for controls vs. 2.5 mg/kg buprenorphine-treated, respectively). Adult brains were collected, immediately dissected into different regions (brain stem, hippocampus, frontal cortex, occipital cortex, parietal cortex, striatum and thalamus), frozen on dry ice and stored at −70°C until binding assays were performed. For brain stem, frontal cortex, occipital cortex and parietal cortex, the tissue from a single brain provided sufficient protein to generate one binding curve. For binding assays using hippocampus, striatum and thalamus, regions from two to three brains were pooled to provide sufficient substrate. Animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health.

Washing procedure to remove residual buprenorphine

After homogenization of rat brain regions using a Polytron, membrane preparations were collected by centrifugation at 20,000 × g and were washed at least five times with 50 mM Tris HCl, pH 7.4. This procedure proved sufficient to remove most of the residual buprenorphine as shown by several lines of evidence (Belcheva et al., 1993).

Opioid receptor binding assays

Rat membrane preparations were assayed for opioid binding activity as described (Belcheva et al., 1993). Membrane preparations (300–800 μg of protein) were incubated in duplicate with 1 nM ³H-U69593 (64 Ci/mmol, Amersham, Arlington Heights, IL) or 1 nM ³H-DAMGE (24–38 Ci/mmol, Chiron) at 25°C for 1 hr, or 2 nM ³H-DPDPE (30 Ci/mmol, Chiron) at 25°C for 3 h. ³H-Deltorphin II (0.5 nM, 55 Ci/mmol, Dupont NEN, Boston MA) binding was performed at 35°C for 45 min. In the ³H-DPDPE experiments, filters were presoaked in incubation buffer containing 0.3% polyethylenimine. B_max and K_d values were estimated from homologous competition binding assays performed in the presence of 10 to 12 different concentrations of the corresponding unlabeled ligand using the LIGAND program (Munson and Rodbard, 1980). In all experiments shown, a single site model fit the data better than a two or more site fit. Statistical analyses of binding data were achieved using the Student’s t test. Protein concentrations were determined by the method of Lowry (Lowry et al., 1951) with bovine serum albumin as a standard.

Cross-linking with ¹²⁵I-β-endorphin

The method developed by Howard et al. 1985 was used with some modifications. Membrane preparations from brain regions of control and buprenorphine treated rats were washed five times with 50 mM Tris-HCl buffer to remove the residual drug. Then the pellets were suspended in 50 mM phosphate buffer, pH 7.4 (50 mM K₂HPO₄, 1 mM EDTA, 10 μM leupeptin and 0.01% bacitracin) and incubated with ¹²⁵I-β-endorphin (2 nM, 2000 Ci/mmol, Chiron) in the presence and absence of 1 μM unlabeled β-endorphin for 1 hr at room temperature. The incubation was halted by centrifugation for 5 min in a centrifuge (Beckman microfuge) at the highest speed. After resuspension of the pellets in phosphate buffer, they were incubated with the cross-linker BSO-COES (1 mM bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, Pierce) for 15 min on ice. The reaction was terminated by addition of 50 mM Tris-HCl, pH 7.4, and centrifugation. To remove labeled β-endorphin that was not cross-linked, samples were resuspended in 50 mM NaCl and incubated for 30 min at room temperature. Samples were adjusted to equal protein content (150–200 μg) and electrophoresed on SDS-PAG (10%). After staining with Coomassie blue to ascertain uniform protein loading, gels were dried and exposed to diagnostic films (Kodak X-Omat, XRP) for a period of 6 to 11 days. The intensity of the bands was quantified with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software. In each experiment samples of total and nonspecific binding for controls and treated animals were run on the same gel. Background was estimated for each lane and subtracted out before determining specific density. Statistical analyses were performed with the JMP program from SAS Institute Inc. (Gary, NC). In representative experiments optical transmission readings of the autoradiograms were also analyzed using a computerized video imaging system (Biological Detection Systems, Inc., Pittsburgh, PA). Background optical transmission as well as nonspecific binding were determined and subtracted from values obtained from various brain regions. Results gained were in good agreement with those obtained using the Phosphorimager.

Results

Effect of buprenorphine on μ opioid density in rat brain regions

Buprenorphine induced down-regulation (30–77%) of μ opioid receptor binding in six of the seven brain regions studied (figs. 1 and 2). In regions such as frontal cortex, thalamus, hippocampus and brain stem, a dose of 0.5 mg/kg buprenorphine was sufficient to elicit down-regulation. Occipital cortex and striatum required a higher concentration of the drug (2.5 mg/kg) to generate similar changes in receptor densities. Parietal cortex μ binding was affected by neither of the two doses of buprenorphine. In frontal cortex and brain stem, 0.5 and 2.5 mg/kg buprenorphine caused a similar reduction in DAMGE binding, although in thalamus down-regulation increased with the higher concentration of the drug. The observed brain-region-specific μ receptor down-regulation was induced by buprenorphine without statistically significant changes in DAMGE affinity.

Fig. 1.

Homologous competition binding curves demonstrating buprenorphine-induced down- and up-regulation of opioid binding in brain regions. In all experiments adult male rats were injected ip with buprenorphine (0.5 or 2.5 mg/kg) or saline 20 hr before they were killed. Homologous competition binding assays were performed on membranes from brain regions and parameters (K_d and B_max) were generated from curves such as these. Data are the mean ± S.E.M. of three experiments.

Fig. 2.

³H-DAMGE (μ) binding changes in brain regions from rats treated with buprenorphine. Corresponding K_d values ranged as follows: 1.1 ± 0.1–1.5 ± 0.1 (controls); 0.7 ± 0.1–2.4 ± 0.6 (0.5 mg/kg buprenorphine) and 0.9 ± 0.3–2.4 ± 0.2 nM (2.5 mg/kg buprenorphine). Numbers in parentheses represent residual binding as % of controls, ^*vs. controls, P < .05. Data are the mean ± S.E.M. of three to six experiments.

Differential effects of buprenorphine on δ₁ and δ₂ opioid binding

Buprenorphine (2.5 mg/kg) up-regulated δ₂-selective deltorphin II binding in frontal and parietal cortices, (fig. 3). There were no δ₂ binding density changes in parietal cortex and striatum after buprenorphine treatment. Consistent with receptor autoradiography studies (Mansour et al., 1987), there was little or no detectable δ binding in hippocampus or thalamus. DPDPE binding to the three cortical regions was unaffected by 0.5 mg/kg buprenorphine treatment (fig. 3). In contrast to deltorphin II binding, 2.5 mg/kg buprenorphine had no effect on DPDPE binding in frontal cortex (B_max =10.2 ± 2.2 [n = 4] and 6.3 ± 0.8 [n = 5] fmol/mg prot. for controls vs. 2.5 mg/kg buprenorphine-treated, respectively). The observed changes in B_max values were not accompanied by modifications in deltorphin II binding affinities.

Fig. 3.

Changes in δ opioid binding to brain regions from buprenorphine-treated rats. δ₁ binding was detected with DPDPE and δ₂ densities with deltorphin II. Corresponding K_d values for deltorphin II ranged from: 1.0 ± 0.05–2.0 ± 0.1 (controls) and 1.0 ± 0.2–1.8 ± 0.3 nM (2.5 mg/kg buprenorphine) and for DPDPE: 1.9 ± 0.6–3.3 ± 1.4 (controls) and 1.6 ± 0.2–2.5 ± 0.6 nM (0.5 mg/kg buprenorphine). Numbers in parentheses represent binding as % of controls, ^*vs. controls, P < .05. Data are the mean ± S.E.M. of three to five experiments.

Up-regulation of κ₁ binding sites in rat brain regions

Buprenorphine administration resulted in the up-regulation (2- to 4-fold) of κ₁-opioid binding in frontal, parietal and occipital cortices and in striatum, but not in brain stem (fig. 4). Although 0.5 mg/kg buprenorphine elevated striatal κ₁-sites, higher doses (≥1.0 mg/kg) of the drug had no effect. U69593 binding sites were not detected in hippocampal membrane preparations. Similar to μ and δ binding, buprenorphine treatment did not evoke changes in U69593 binding affinity in the brain regions in most cases examined.

Fig. 4.

³H-U69593 (κ₁) binding densities in brain regions from rats administered buprenorphine. Corresponding K_d values ranged as follows: 1.1 ± 0.2–4.8 ± 0.9 (controls); 2.2 ± 0.3–5.7 ± 1.2 (0.5 mg/kg buprenorphine); 2.2 ± 0.6 (1.0 mg/kg buprenorphine) and 2.2 ± 0.6–5.3 ± 0.4 nM (2.5 mg/kg buprenorphine). Numbers in parentheses represent binding as % of controls, ^*vs. controls, P < .05. ^#vs. treated (0.5 mg/kg), P < .05. Data are the mean ± S.E.M. of three to six experiments.

¹²⁵I-β-endorphin cross-linking of membrane preparations from rat brain regions

Electrophoretic analysis of ¹²⁵I-β-endorphin cross-linked membrane preparations from frontal, parietal and occipital cortices of control and buprenorphine (2.5 mg/kg) treated rats revealed the existence of two major bands at 60- to 65- and 43- to 46-kDa (which includes the contribution of β-endorphin). The reduction in intensity of the reaction products in the presence of 1μM β-endorphin suggests that they are related to opioid binding sites (data not shown). Buprenorphine treatment differentially affected the intensity of the reaction products (fig. 5). The quantification of the gels shows a decrease in the labeling of the 60- to 65-kDa band and no significant effect on the 43- to 46-kDa band in frontal cortex. A significant decrease in the intensity of both bands was seen for occipital cortex upon buprenorphine treatment. In contrast, no effect was observed for the 60- to 65-kDa reaction product in parietal cortex membranes but an increase in the 43- to 46-kDa band was found.

Fig. 5.

Densitometric analysis of ¹²⁵I-β-endorphin cross-linked bands in frontal, parietal and occipital cortex membrane preparations from rats treated with buprenorphine. Rats were administered (i.p.) 2.5 mg/kg buprenorphine; 20 hr later they were sacrificed and their brains were removed and dissected. Membrane preparations (after five washes with 50 mM Tris-HCI, pH 7.4) were cross-linked with 2 nM ¹²⁵I-β-endorphin in the presence (nonspecific binding) and absence (total binding) of 1 μM unlabeled β-endorphin as described in “Materials and Methods.” Equal amounts of protein (150–200 μg) were solubilized in sodium dodecyl sulfate (SDS) buffer and polyacrylamide gel ectrophoresis (PAGE) (10%) was performed. Specific densities of the 60- to 65- and 43- to 46-kDa bands in each brain region were determined as the difference between the density of the total and nonspecific binding for control and buprenorphine treated samples. Observed density changes induced by the buprenorphine treatment are expressed as percent of control values that were normalized to 100%. Data are the mean ± S.E.M. of three to four experiments, ^*vs. controls, P < .05.

Discussion

In a previous study, buprenorphine administration to rats was shown to induce down-regulation of μ. and up-regulation of κ₁ and δ₂ receptors in forebrain (Belcheva et al., 1993). Our present results indicate that the buprenorphine-engendered differential receptor adaptation is brain-region specific. Down-regulation of μ opioid binding was found in all brain regions analyzed except parietal cortex. Apparent loss in sites due to receptor blockade by residual buprenorphine was ruled out by several lines of evidence (Belcheva et al., 1993), including the absence of elevated ³H-DAMGE K_d values for membranes from treated animals. This was important to ascertain given the fact that buprenorphine dissociation from the opioid receptor is slow (for a review see Rothman et al., 1995). Previously ³H-DSLET was used to measure δ₂ binding but this peptide also binds to μ, and possibly other receptors. In this study, we used the more δ₂ selective ligand deltorphin II and an increase in binding densities was detected in two of four regions examined. In contrast, treatment with 0.5 to 2.5 mg/kg buprenorphine did not alter δ₁ opioid binding in any brain regions tested. A 2- to 4-fold up-regulation of κ₁ opioid binding in parietal and occipital cortices as well as striatum, with no change in brain stem, were observed after buprenorphine administration.

Earlier behavioral investigations suggested that buprenorphine acts as a partial agonist (Cowan et al., 1977; Martin et al., 1976). Subsequently, μ and possibly δ and κ agonist activity of buprenorphine were reported in a number of other pharmacological studies (Dum and Herz, 1981; Sadee et al., 1982; Kajiwara et al., 1986). However, with the availability of more κ-selective ligands as competitors, buprenorphine has proven to be a potent κ opioid receptor antagonist (Richards and Sadee, 1985; Leander, 1987; Negus et al., 1989). The up-regulation data generated in this and our previous publication on buprenorphine (Belcheva et al., 1993) support the notion that buprenorphine is also a δ₂ antagonist. Moreover, independent evidence that buprenorphine is a δ₂ antagonist has originated from recent studies with a cloned δ opioid receptor containing a point mutation at residue 95 (Kong et al., 1993). The aspartate at position 95, which is in the second transmembrane domain of this G protein coupled receptor, is known to be critical for high affinity δ binding. A mutant which has an asparagine in that position (D95N) binds with lowered affinity to six δ agonists tested. However, the K_d of buprenorphine for the D95N mutant did not vary from that of the wild-type as seen for antagonists such as naltrindole and its benzofuran analog. As a result of these properties some authors have referred to buprenorphine as a mixed agonist-antagonist (Rothman et al., 1995).

The ¹²⁵I-β-endorphin cross-linking data (fig. 5) further support the evidence for buprenorphine-induced down-regulation obtained in homologous competition studies with ³H-DAMGE. For example, in frontal and occipital cortex membranes the labeling of the 60- to 65-kDa band was significantly reduced, although the intensity of this band was not changed in parietal cortex membranes. The labeling of the 43- to 46-kDa band is attenuated differentially by buprenorphine treatment. The identity of the peptide responsible for this band is unknown. It may be either a truncated form of opioid receptor (which could be derived from μ, or δ types as suggested by opioid receptor purification studies (Bidlack et al., 1981; Fujioka et al., 1985; Loukas et al., 1994) and/or a completely different molecule that is associated with the receptor and cross-linked to β-endorphin because of its proximity. Nevertheless, these data demonstrate that both 60- to 65- and 43- to 46-kDa bands can be diminished upon agonist-induced down-regulation of opioid receptors.

The differential receptor subtype and brain region-specific up- and down-regulation may be explained by several phenomena. Some brain regions may receive more drug secondary to increased blood flow and/or increased blood brain barrier permeability. Although this possibility cannot be ruled out, other data argue against it. For example, receptor autoradiography of naltrexone-induced opioid sites has revealed brain subregion-specific up-regulation of μ, opioid receptors in neocortex, septum, amygdala, substantia nigra and thalamus as discussed above (Tempel et al., 1984; Paden et al., 1987). Moreover, we observed extensive down-regulation of μ binding in brain stem but no κ₁ up-regulation in response to buprenorphine. Similarly, parietal cortex κ₁ receptors are up-regulated but μ receptors are unaffected by the buprenorphine regimen.

A second explanation for the regional specificity is the existence of time- and dose-dependency differences for receptor adaptation in various brain regions. Naltrexone induced up-regulation of δ sites in some rat brain regions in 6 hr, although in others >24 hr were required (Belcheva et al., 1994). In addition, chronic etorphine treatment of rats produced a time-dependent decrease in diprenorphine binding to membranes of different brain regions. In striatum and cortex, 1-day etorphine treatment did not alter opioid binding, although a significant decrease in binding was found in mid-brain membrane preparations. After 3 days of etorphine exposure, all three brain regions showed opioid receptor down-regulation (Tao et al., 1987). Moreover, μ opioid binding was increased in many rat brain regions after chronic (7 days) administration of a low dose of naloxone (0.5 mg/kg/h), although δ sites measured with DADLE and κ sites labeled with bremazocine were not significantly affected. In contrast, 3 mg/kg/hr naloxone caused an up-regulation of all three opioid receptor types, suggesting that this phenomenon takes place when a sufficient proportion of the receptors in vivo are occupied by the ligand (Morris et al., 1988).

Finally, we have shown that δ₂ but not δ₁ receptors undergo up-regulation. Interestingly, δ₂ but not δ₁ opioid receptor subtypes were implicated in the development of physical dependence on morphine in mice (Miyamoto et al., 1994). Thus, opioid receptor adaptation may be subtype-specific and dependent upon its physiological role, as has been proposed for β-adrenergic receptors (Liggett et al., 1993; Barak et al., 1994). Although β₂ adrenergic receptors undergo agonist-induced down-regulation and sequestration, β₃ does not; this was reported to be consistent with its function in adipocytes which is considered to be critical for a sustained, tonic output of energy substrates for extended elevated sympathetic activity. In addition, in contrast to β₂, the β₃ receptor lacks two determinants, tyrosine residues and a sequence (NPXXY) in its C-terminal close to the end of the seventh transmembrane domain, that are believed to be among the important signals for internalization and sequestration, respectively (Liggett et al., 1993; Barak et al., 1994). All opioid receptors cloned thus far have the (NPXXY) sequestration sequence, but not the internalization signal (Evans et al., 1992; Kieffer et al., 1992). However, we cannot exclude the possibility that opioid receptors may have a different signaling sequence for down-regulation or that yet-to-be-cloned opioid receptors contain C-terminal tyrosines.

Whether buprenorphine-induced differential up- and down-regulation in rat brain regions has any relevance to the utility of this drug in treating dual cocaine and heroin abuse (Mello and Mendelson, 1980; Kosten et al., 1989; Johnson et al., 1992) is contingent on a greater understanding of the underlying mechanisms of drug dependence and tolerance. Nevertheless, a recent study on differential regulation of brain opioid receptors after repeated cocaine administration to rats showed significant increases in ³H-DAMGE binding measured in some cortex regions, nucleus accumbens, caudate putamen and basolateral amygdaloid nucleus of cocaine-treated animals (Unterwald et al., 1992). Chronic cocaine exposure of rats also differentially altered ³H-naloxone binding to critical brain reward regions in investigations utilizing receptor autoradiography (Hammer, 1989). An increase in opioid receptor labeling was found for nucleus accumbens and the regions to which it projects, although the receptor density was reduced in regions containing neurons of ascending catecholamine systems. Based on these and our results on the differential effects of buprenorphine, an attractive hypothesis is that, by down-regulating μ and up-regulating some δ and κ opioid receptors in rat brain, buprenorphine may be capable of suppressing the effect of cocaine and other drugs of abuse.

ABBREVIATIONS

BrS

brain stem

DAMGE

[D-Ala²,N-mephe⁴Gly-ol⁵] enkephalin

Deltorphin II

[Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH₂]

DPDPE

[D-Pen²D-Pen⁵] enkephalin

DSLET

[D-Ser²[scaP]L-Leu⁵] enkephalyl-Thr

FrC

frontal cortex

Hip

hippocampus

OcC

occipital cortex

PrC

parietal cortex

Str

striatum

Tha

thalamus