Identification of Roughskin Newt Medullary Vasotocin Target Neurons with a Fluorescent Vasotocin Conjugate CHRISTINE M. LEWIS, 1,2 E. KURT DOLENCE, 2,3 CATHERINE S. HUBBARD, 1,2 AND JAMES D. ROSE 1,2 * 1 Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming 82071 2 Neuroscience Program, University of Wyoming, Laramie, Wyoming 82071 3 School of Pharmacy, University of Wyoming, Laramie, Wyoming 82071 ABSTRACT Arginine 8 vasotocin (AVT), a neurohypophyseal peptide in nonmammalian vertebrates, plays a key role in the regulation of social behaviors related to reproduction. In male roughskin newts (Taricha granulosa), AVT is an important facilitator of several reproductive behaviors, including courtship clasping of females. Although AVT is known to act in certain brain regions and AVT receptors have been localized to some extent, specific target neurons for this peptide have not been identified in any species. Internalization of a receptor-specific conjugate of AVT and the fluorescent dye Oregon green was used to identify AVT target cells in the medulla of male roughskin newts. Medullary neurons are of interest because they appear to mediate facilitation of clasping by AVT. Direct application of AVT-Oregon green to the fourth ventricular surface of the medulla in vivo resulted in conjugate internalization by a widespread population of medullary neurons, particularly in the medial reticular formation and nuclei of cranial nerves V, VII, VIII, IX, and X. Some fourth-ventricle ependymal cells were also labeled. Reticulospinal neurons, which play an important role in clasping, were identified by retrograde labeling with tetrameth- ylrhodamine dextran amine. AVT-Oregon green was internalized by 72% of these neurons. These results show that AVT can directly affect a very large and diverse medullary neuronal population, which may underlie the peptide’s role in multiple neuroendocrinological processes, including autonomic and behavioral regulation. Selectivity of the AVT action may arise through interac- tions between AVT and steroids such as corticosterone. J. Comp. Neurol. 491:381–389, 2005. © 2005 Wiley-Liss, Inc. Indexing terms: reticular formation; reticulospinal; neuropeptide internalization; sexual behavior; clasping; urodele Arginine vasotocin (AVT), a neurohypophyseal hor- mone in nonmammalian vertebrates, and arginine va- sopressin (AVP), the mammalian homologue to vasoto- cin, influence a variety of behaviors across a wide range of vertebrate species. These neuropeptides are impor- tant for regulation and expression of social and repro- ductive behaviors, especially those allowing discrimina- tion of, and interaction between, conspecifics. Information about the specific neuronal populations me- diating the action of these neuropeptides comes largely from studies employing localized infusions. What is clearly lacking is a demonstration of the actual target neurons that transduce the neurobehavioral effects of AVT and AVP. So far, this question has been ap- proached by identification of receptors or binding sites for these neuropeptides or related ligands. The V 1a -type receptor is the predominant AVP receptor in the mammalian brain (Jard et al., 1987). This receptor Grant sponsor: National Institutes of Health (NIH)–National Center for Research Resources; Grant number: P20 RR015553 (to J.D.R.); Grant sponsor: University of Wyoming Center of Biomedical Research Excellence; Grant sponsor: NIH; Grant number: GM062139 (to E.K.D); Grant sponsor: University of Wyoming Center of Biomedical Research Excellence Micros- copy Core. *Correspondence to: James D. Rose, Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071. E-mail: trout@uwyo.edu Received 24 January 2005; Revised revised 16 May 2005; Accepted accepted 7 June 2005 DOI 10.1002/cne.20701 Published online in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 491:381–389 (2005) © 2005 WILEY-LISS, INC.