A new oxytocin-saporin cytotoxin for lesioning oxytocin-receptive neurons in the rat hindbrain

Abstract

Evidence suggests that release of oxytocin in the nucleus tractus solitarius (NTS) of the hindbrain from descending projections that originate in the paraventricular nucleus can inhibit food intake by amplifying the satiety response to cholecystokinin (CCK). To further evaluate this mechanism in rats, we used a novel cytotoxin, saporin conjugated to oxytocin (OXY-SAP), a compound designed to destroy cells that express oxytocin receptors (OXYr). OXY-SAP was injected directly into the NTS to lesion neurons that express OXYr and that are implicated in potentiating CCK's satiety effects. The control consisted of injection of saporin conjugated to a nonsense peptide. We found that OXY-SAP was cytotoxic to human uterine smooth muscle cells in vitro, demonstrating that OXY-SAP can lesion cells that express OXYr. Using laser capture microdissection and real-time quantitative PCR, we demonstrated that OXYr mRNA levels were reduced in the NTS after OXY-SAP administration. Moreover, we found that OXY-SAP attenuated the efficacy of CCK-8 to reduce food intake and blocked the actions of an OXYr antagonist to stimulate food intake. The findings suggest that OXY-SAP is an effective neurotoxin for in vivo elimination of cells that express OXYr and is potentially useful for studies to analyze central nervous system mechanisms that involve the action of oxytocin on food intake and other physiological processes.

Figure 1

Cytotoxic effect of OXY-SAP on human uterine myometrial cells after 72 h in culture. Measurements of cell counts were made on four to six culture wells for each treatment condition. Data represent means ± sem. *, P < 0.05 vs. media (no saporin).

Figure 2

Effect of NTS injection of OXY-SAP on OXYr mRNA levels in NTS, compared with NTS injected with CON-SAP. Graph shows that OXY-SAP reduced OXYr mRNA expression in NTS (n = 4–9/group). Data represent means ± sem. *, P < 0.05.

Figure 3

Histological verification and distribution of injection sites in mNTS. Representative photomicrographs taken of an injection site from an OXY-SAP (A) and a CON-SAP-treated animal (B). The location of the injection site is centered in the mNTS, rostral to the AP. Coronal diagrams of the rat brain based on the atlas of Paxinos and Watson (22) show the anatomical distribution of injection sites for groups of animals with cannulae aimed at the mNTS (C). The solid circles indicate sites of injections in the medial NTS. 4V, Fourth ventricle.

Figure 4

Effect of 3V administration of the OXYr antagonist [d(CH(₂)₅¹,Try (Me)², Orn⁸)]-oxytocin on food intake in OXY-SAP and CON-SAP-treated animals. Graph shows that NTS administration of OXY-SAP eliminated the stimulation of food intake in response to 3V administration of the OXYr antagonist (10 μg per 2 μl) (n = 4–10/group). Data represent means ± sem. **, 0.05 less than P < 0.1 vs. vehicle; †, P < 0.01 vs. vehicle.