Immunoreactive neurons in the brain of two mouse strains after incubation with an antiserum recognizing Asp-Val-Val-Gly.NH 2 (DVVG), the C-terminal fragment of (D-Ala 2 )-deltorphin I Loredana D’Este a, *, Arianna Casini a , Stefano Puglisi-Allegra b , Simona Cabib b , Ikuo Tooyama c , Hiroshi Kimura c , Tindaro G. Renda a a Department of Human Anatomy, University ‘La Sapienza’, via A.Borelli 50, 00161 Rome, Italy b Department of Psychology, Neuroscience Section, University ‘La Sapienza’ and IRCCS Fondazione Santa Lucia, Rome, Italy c Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu, Japan Received 13 June 2001; received in revised form 14 February 2002; accepted 27 May 2002 Abstract D-Ala 2 -deltorphin I (DADTI) is a heptapeptide amide first extracted from frog skin that displays a high selectivity and affinity for delta opioid receptors. Previous studies using a polyclonal antiserum specific for its C-terminal tetrapeptide-amide (DVVG) have already described in rat and mouse brain the presence of immunoreactive neurons, most of them belonging to the mesencephalic dopaminergic neurons. C57BL/6J (C57) and DBA/2J (DBA) are two inbred strains of mice well known for showing marked genotype-dependent differences for phenotypes related to differential brain dopamine functioning. Brain specimens of both inbred mouse strains were frozen, cut and immunostained using the same antiserum. Some sections were also double immunostained with monoclonal anti-tyrosine hydroxylase (TH). DVVG-immunoreactive neurons were observed among both dopaminergic and non- dopaminergic neurons. DVVG- and TH-immunoreactive neurons were observed among the dopaminergic A8, A9 and A10 mesencephalic nuclei. They were on average 21.9% more numerous in DBA than in C57 mice. DVVG-immunoreactive nerve fibres could be seen in limbic, striatal, cortical and thalamic areas. The distribution patterns of DVVG-IR and TH-IR nerve fibres differed most conspicuously within the infralimbic, prelimbic and cingulate cortices, forming a dense network in DBA but rare in C57 mice. Non-dopaminergic DVVG-immunoreactive neurons did not differ significantly in the two strains. Our finding that the number and distribution pattern of this dopaminergic neuronal subpopulation differed in the two mouse strains could provide morphological support for the known behavioural differences between the DBA and C57 strains under normal and experimental conditions. # 2002 Elsevier Science B.V. All rights reserved. Keywords: C57BL/6J; DBA/2J; Amphibian skin peptides; Opioid peptides; Dopaminergic pathways; Immunohistochemistry; Tyrosine hydroxylase 1. Introduction (D-Ala 2 )deltorphin-I (DADTI), is a naturally occur- ring heptapeptide amide (Tyr-D-Ala-Phe-Asp-Val-Val- Gly.NH 2 ) originally extracted from the skin of the South American frog Phyllomedusa bicolor together with D-Ala 2 -deltorphin II (DADTII). DADTII differs from DADTI only in the substitution of a glutamate residue (Glu) in position 4. Both heptapeptides belong to a larger amphibian skin heptapeptide amide family that includes dermorphins, extracted from the skin of Phyllomedusa sauvagei (a review in Erspamer, 1992). All these peptides have high affinity and selectivity for opioid receptors: dermorphins for mu opioid and deltorphins for delta opioid receptors (Dupin et al., 1991; Gouarderes et al., 1993; Renda et al., 1993). Pharmacological studies have shown that deltorphins, when injected into the rat cerebral lateral ventricle, induce locomotory stereotypies (i.e. grooming, sniffing Abbreviations: A8, retrorubral field; A9, substantia nigra; A10, ventral tegmental area; C57, C57BL/6J inbred strain; DADTI, (D- Ala 2 )-deltorphin I; DBA, DBA/2J inbred strain; DVVG, C-terminal tetrapeptide amide of DADTI: Asp-Val-Val-Gly.NH 2 ; TH, tyrosine hydroxylase. * Corresponding author. Tel.:  /39-06-490-125; fax:  /39-06-445- 2349 E-mail address: loredana.deste@uniroma1.it (L. D’Este). Journal of Chemical Neuroanatomy 24 (2002) 189  /198 www.elsevier.com/locate/jchemneu 0891-0618/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0891-0618(02)00054-6