Between-group variations in count ideals for each region were tested for statistical significance by using ANOVA, with treatment condition (i.e., NST toxin or NST vehicle plus intraperitoneal CCK or intraperitoneal vehicle) mainly because the independent variable. treatment, whereas CCK-induced neural activation in the parabrachial nucleus and amygdala appeared normal. These findings suggest that hindbrain NA neurons are an integral component of brainstem circuits that mediate CCK-induced anorexia and also are necessary for hypothalamic but not parabrachial or amygdala reactions to gastric sensory activation. All methods conformed to National Institutes of Health guidelines and were authorized by the University or college of Pittsburgh Animal Care and Use Committee. Data from 20 adult male Sprague Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) are included in this report. Rats were singly housed with access to water and pelleted rat chow (Purina, St. Louis, MO) except during Tirofiban Hydrochloride Hydrate food intake experiments. Colony rooms were managed at 22-23C and kept on a 12 hr light/dark cycle, with lamps on from 7:00 A.M. to 7:00 P.M. Rats weighed 180-225 gm at the time of toxin injection surgery treatment and 200-325 gm at the time of food intake experiments and the terminal cFos study. Rats received bilateral injections of anti-DbH-saporin toxin (toxin; = 10; Advanced Targeting Systems, San Diego, CA) or 0.15 m NaCl (vehicle; = 10) into the caudal NST. Each rat was used in a food intake experiment and in a terminal cFos study. Toxin or vehicle microinjections were made into the NST at six sites (Fig. 1). Injection sites encompassed the NST region that contains the highest incidence of cFos manifestation by DbH-positive neurons after CCK treatment, related to the location of the A2 cell group within the caudal medial NST (Sawchenko and Swanson, 1982; Rinaman et al., 1993; Rinaman et al., 1995). Rats were anesthetized with halothane (2-5% in 100% oxygen) and secured inside a stereotaxic framework using blunt ear bars, Tirofiban Hydrochloride Hydrate with the head ventroflexed. The skin on the dorsal neck surface was shaved and incised, and the neck muscles were retracted and bluntly dissected to expose the meninges overlying the dorsal surface of the caudal medulla. With Rabbit Polyclonal to CSTL1 the aid of a medical microscope, the meninges were cut having a sterile needle to uncover the AP. Under visual guidance, a glass micropipette tip (outer diameter of 50-75 m) filled with toxin or vehicle and affixed to a 0.5 l Hamilton syringe was positioned on the midline in the caudal limit of the AP and was then moved 0.25 mm lateral and 0.5 mm below the medullary surface for the first, most caudal injection site. The second injection site was located 0.25 mm below the lateral border of the AP at its midrostrocaudal extent, and the third site was located 0.25 mm below the lateral border of the AP at its most rostral extent (Fig. 1). The three NST injection sites were then duplicated contralaterally. At each injection site, 50 nl of sterile 0.15 m NaCl vehicle containing 0 or 5 ng of toxin was delivered by manual pressure injection over 30 sec. Toxin Tirofiban Hydrochloride Hydrate was freshly prepared from a freezing stock answer within Tirofiban Hydrochloride Hydrate 2 hr of injection. Results from a published statement (Madden et al., 1999) and additional pilot studies confirmed that this concentration and volume of toxin damaged the majority of DbH-positive neurons within the caudal NST (observe Results). Lower toxin doses were less effective, whereas higher doses produced marked cells necrosis. After each microinjection, the pipette tip was left in place for 1-2 min and then withdrawn. The skin incision was closed with stainless steel clips after the final injection. Rats were returned to their home cages after recovery from anesthesia. Open in a separate window Number 1. Schematic representation of NST sites (X) targeted for 50 nl microinjections of toxin or vehicle. Related contralateral sites were targeted in each rat. Schematics adapted from commercially available digital documents (Paxinos and Watson, 1998). CC, Central canal; py, pyramidal tract; sol, solitary tract. The fact that saporin toxin microinjected near NA cell body efficiently lesions those cells provides empirical evidence for vesicular launch and concomitant extracellular exposure of DbH within the toxin injection site (Madden et al., 1999)..