Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9635-9639, October 1993 Pharmacology [D-Ala2]Deltorphin I-like immunoreactivity in the adult rat brain: Immunohistochemical localization (amphibian skin peptides/&opioid receptors/dopamine/calbindin) IKUO TOOYAMA*, HIROMICHI ABE*t, TINDARO RENDAt, VITTORIO ERSPAMER§, AND HIROSHI KIMURA*$ *Institute of Molecular Neurobiology and tDepartment of Ophthalmology, Shiga University of Medical Science, Otsu, 520-21 Japan; and Institutes of tHuman Anatomy and Medical Pharmacology, University "La Sapienza," 00161 Rome, Italy Contributed by Vittorio Erspamer, July 7, 1993 ABSTRACT Using a specific antiserum recently raised comotor activity, sniffing, rearing, grooming (10), as well as against [D-Ala2]deltorphin I (DADTI: Tyr-D-Ala-Phe-Asp-Val- facilitation of social contacts (11) and memory consolidation Val-Gly-NH2), a highly selective ligand for 6-opioid receptors, (12). we have previously demonstrated the occurrence of positive We have recently produced a polyclonal anti-DADTI se- immunostaining in several structures of mouse brain. We rum, which specifically recognizes its carboxyl-terminal tet- describe here the neuroanatomical distribution patterns of rapeptide region (13). Immunohistochemical studies of DADTI-immunoreactive neuronal bodies, axons, and tany- mouse brain have detected DADTI-immunoreactive (IR) cytes in rat brain. Positive neuronal somata were localized neuronal cells in the midbrain tegmentum and positive nerve mainly in the ventral mesencephalon, including the ventral fibers in the accessory olfactory bulb and neostriatum. tegmental area and the pars compacta of the substantia nigra. This study was undertaken to elucidate the neuroanatom- A minor population of positive somata was found in the pars ical distribution patterns of DADTI immunoreactivity in the reticulata and pars lateralis of the substantia nigra, raphe rat brain. nuclei, supramammillary nucleus, and retrorubral reticular nucleus. All these regions, except for the supramammillary nucleus, contain dopamine cell bodies. Intensely stained posi- MATERIALS AND METHODS tive nerve fibers could be traced along the medial forebrain Production and Characterization of DADTI Antiserum. The bundle. Dense positive terminals were seen in the neostriatum, DADTI antiserum was raised in the rabbit as described by nucleus accumbens shell, olfactory tubercle, septal areas, Abe et al. (13) using as antigen synthetic DADTI conjugated cingulate, and medial prefrontal cortex. Double-immunostain- to poly-L-glutamate with water-soluble carbodiimide. Immu- ing study revealed that, in the substantia nigra, almost all nospot and immunoabsorption assays designed to test spec- (97.8%) DADTI-positive neurons colocalized with tyrosine ificity of the antiserum showed that it reacted specifically hydroxylase (TH), and the doubly stained cells occupied about with the amidated carboxyl-terminal pentapeptide: Phe-Asp- one-third (29.1%) of the total population of TH-positive neu- Val-Val-Gly-NH2. The antiserum, therefore, possessed a rons. Only a few DADTI/TH-positive cells also stained for marked cross-reactivity not only with the [L-Ala2]DADTI 28-kDa calbindin D, although many neurons double-stained for stereoisomer but also with DADTI analogs having the D-ala- 28-kDa calbindin D and TH. In contrast, the supramammillary nine residue substituted by D-leucine, D-aspartate, glycine, nucleus contained a number of DADTI-positive cells, which aminobutyric acid, or sarcosine. The antiserum did not nearly always stained positively for 28-kDa calbindin D but did cross-react with (i) DADTI analogs shortened at the carboxyl not stain for TH. The association of DADTI-like immunore- terminus, (ii) deamidated DADTI, (iii) DADTI analogs hav- activity with certain dopaminergic pathways seems of partic- ing the Asp4 residue substituted by glutamate (as in ular interest. A small population of DADTI-immunostained [D-Ala2]deltorphin II) or D-aspartate, asparagine, or alanine, tanycytes was present in the ventral part of the third ventricle and (iv) Phyllomedusa sauvagei deltorphin (Tyr-D-Met-Phe- wall. His-Leu-Met-Asp-NH2). Similarly, no cross-reaction was observed with dermorphins or with any of the classical [D-Ala2]Deltorphin I (DADTI), an opioid peptide recently mammalian opioid peptides or other common brain neu- isolated from the skin of the South American frog Phyllo- ropeptides. The DADTI sequence has little homology with medusa bicolor, displays high affinity and selectivity for known peptides or proteins other than those of the dermor- 8-opioid receptors (1, 2). cDNAs encoding deltorphin pre- phin/deltorphin family. cursors have been cloned from the skin of the same amphib- Tissue Preparation. Adult male Wistar rats, weighing 200- ian species (3). The precursors contained three DADTI 250 g, were used for the study. Under deep anesthesia with sequences and only a single [D-Ala2]deltorphin II sequence. Nembutal (50 mg/kg, i.p., Abbott), each animal was perfused Because its L-isomer is pharmacologically inactive (1), D-ala- via the left ventricle with 0.01 M phosphate-buffered saline nine or at least a D-amino acid in the second position appears (pH 7.4), followed by an ice-cold fixative containing 4% essential to its opiate-like activity. In turn, the carboxyl- (wt/vol) paraformaldehyde, 0.2% picric acid, and 0.35% terminal region of deltorphins seems crucial for addressing glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The brain the molecules toward the 6-type receptor (4-8). Recently, was quickly removed from the skull and sliced into 5- to 6-mm DADTI-binding sites have been demonstrated by autoradi- coronal blocks, which were postfixed for 2 days in a post- ography with either 125I-labeled DADTI in mouse brain (9) or fixative containing 4% paraformaldehyde and 0.2% picric [3H]DADTI in rat brain (unpublished work). DADTI or acid in 0.1 M phosphate buffer at 4°C. After cryoprotection [D-Ala2]deltorphin II injected into the rat cerebral ventricle by placing the tissue for 2 days in 0.1 M phosphate buffer/ induces several central effects, including stimulation of lo- 15% sucrose/0.1 M sodium azide at 4°C, each block was cut The publication costs of this article were defrayed in part by page charge Abbreviations: DADTI, [D-Ala2]deltorphin I; TH, tyrosine hydroxy- payment. This article must therefore be hereby marked "advertisement" lase; CaBP, 28-kDa calbindin D; IR, immunoreactive. in accordance with 18 U.S.C. §1734 solely to indicate this fact. $To whom reprint requests should be addressed. 9635 Downloaded by guest on September 23, 2021 9636 Pharmacology: Tooyama et al. Proc. Natl. Acad. Sci. USA 90 (1993) into 20-,um-thick coronal sections in a cryostat (Yamato, for 1-2 days at 4°C. The antibody binding was detected Tochigi, Japan). The sections were rinsed for at least 4 days similarly to the first cycle, except that nickel ammonium with several changes of 0.1 M phosphate-buffered saline/ sulfate was eliminated from the 3,3'-diaminobenzidine solu- 0.3% Triton-X 100 (PBST). To improve antibody penetration, tion, yielding a yellow-brown reaction product. some sections were treated with protease papain (0.1 inter- national unit per ml of PBST, 10 min at 37°C). Immunohistochemical Procedure. Free-floating sections RESULTS were incubated for 2-3 days at 4°C with the DADTI antiserum (diluted 1:5000-10,000), for 1 hr at room temperature with DADTI immunoreactivity was observed in neurons and biotinylated anti-rabbit IgG (diluted 1:1000, Vector), and for tanycytes. Fig. 1 presents maps of DADTI-IR neuronal 1 hr at room temperature with avidin-biotin-peroxidase somata and their processes in seven schematic drawings of complex (diluted 1:4000, ABC Elite, Vector). All sera were coronal sections, with reference to the atlas of Paxinos and diluted with PBST, and sections were always rinsed in PBST Watson (15). Typical examples are shown with micrographs after each step. Peroxidase activity was revealed by 0.02% in Figs. 2 and 3. 3,3'-diaminobenzidine (Wakenyaku, Kyoto, Japan) in 0.05 M Localization ofDADTI-IR Cell Bodies. DADTI-IR neuronal Tris HCl buffer, pH 7.6/0.005% H202/0.3% nickel ammo- somata were localized mainly in the ventral part of the nium sulfate. The stained sections were mounted on glass midbrain tegmentum, including the ventral tegmental area slides, dehydrated, and cleared; coverslips were applied with and the pars compacta of the substantia nigra (Figs. 1 and 2 Entellan. Control experiments included the omission of the A and B). In the pars compacta, positive somata were primary antiserum or its substitution with either buffer or distributed throughout the rostrocaudal extent of the struc- preimmune antiserum or preabsorbed serum (13). None of ture, but in a coronal plane they were almost always confined the controls showed specific staining. to the ventral tier (Fig. 2A). Positive cell bodies were also Sections were double-immunostained for DADTI and ty- scattered in the supramammillary nucleus (Fig. 1E), the rosine hydroxylase (TH) or 28-kDa calbindin D (CaBP), retrorubral reticular nucleus (so-called A8 region, Fig. 1G), according to Akiyama et al. (14). In brief, after completion of the caudal linear nucleus ofraphe (Fig. 1G), the rostroventral the first 3,3'-diaminobenzidine/nickel reaction for DADTI, part of the raphe dorsalis (Fig. .G), and both the pars yielding a blue-purple reaction product, sections were treated reticulata and the pars lateralis of the substantia nigra (Fig. with 0.5% H202/PBST for 30 min to destroy any residual 2A). Around these positive cells, many dendritic or axonal peroxidase activity. Sections were then incubated for the processes were clearly observed branching into the pars second cycle with mouse monoclonal antibodies to TH reticularis (Fig. 2B). Most positive axons projected medially (diluted 1:40,000, Chemicon) or CaBP (diluted 1:5000, Sigma) to join the medial forebrain bundle (Fig. 1 C and D). A BC Co FIG. 1. Schematic mapping of DADTI-IR cells (0) and fibers (-).