Fluorescent Vasotocin Conjugate for Identification of the Target Cells for Brain Actions of Vasotocin

Abstract

The effects of neuropeptides on the brain are a major focus of neuroendocrine research, and little progress has been made in the identification of the target neurons for many neuropeptides. Arginine8-vasotocin (AVT) is a neurohypophyseal peptide present in nonmammalian vertebrates that controls many neural and behavioral functions. Here we describe synthesis and functional characterization of an AVT-Oregon green conjugate 1 (AVT-OG 1) that can be used in vivo to identify AVT target neurons. Application of AVT-OG 1 to the brainstem of an amphibian produces rapid, endosome-like internalization together with typical AVT-like neurophysiological effects. Thus, preparation of AVT-OG 1, which preserves the peptide's neurophysiological effects, is useful as a fluorescent marker for AVT target neurons. Consequently, AVT-OG 1 conjugate will have considerable utility for analyzing the neural actions of AVT in the intact brain.

Brainstem reticulospinal neurons are targets for corticotropin-releasing factor-Induced locomotion in roughskin newts

“…An endosome was defined as a spot of fluorescence greater than 0.1 µm but less than 0.5 µm in diameter. These values are consistent with other studies in which endosomes have been counted for quantification purposes (Lewis et al, 2004; Lutz et al, 1991; Hubbard et al, 2009). Cell counting was accomplished by overlaying each image with a 300 × 300 µm grid directly over the midline of the tissue section using Adobe Photoshop (v. 6.0).…”

Identification of brain target neurons using a fluorescent conjugate of corticotropin-releasing factor

“…Lastly, we verified the functional efficacy of CRF-TAMRA 1 by showing that the conjugate retains its typical behavior-activating effects in vivo . This combination of in vivo neurohistological and behavioral testing of the conjugate's functional properties was similar to that used in our recent development of a fluorescent conjugate of the peptide arginine vasotocin (AVT-OG 1 ; Lewis et al, 2004; Lewis et al, 2005). …”

“…Surgical procedures were similar to those routinely used in this laboratory (Lowry et al, 1996; Lewis et al, 2004). Following anesthetization by immersion in 0.1% MS-222 (tricaine methanesulfonate, Sigma-Aldrich, E10521), cellulose tissue soaked in anesthetic was draped over the newt's body to maintain anesthesia and facilitate in transcutaneous respiration during surgery.…”

Localization of three types of arginine vasotocin receptors in the brain and pituitary of the newt Cynops pyrrhogaster

“…In our study, we have histochemically confirmed the presence of V1a-type receptor in these regions of the brain of C. pyrrhogaster. Medullary AVT target neurons, particularly those in the medial reticular formation and nuclei of cranial nerves, have recently been visualized by means of Oregongreen-conjugated AVT (Lewis et al 2004(Lewis et al , 2005. These neurons coincide with those exhibiting V1a-type-receptorlike immunoreactivity in the brain of C. pyrrhogaster.…”