Procedures used to label three nerves with fluorescent tracers were similar to those described by May and Hill (2006) (link) and Mangold and Hill (2007) (link). Specifically, the CT, GSP, and IX nerves were labeled with anterograde tracers to determine the volume and spatial organization among the afferent nerve terminal fields in the rostral NTS during postnatal development. The density of the label was not quantified because of inaccuracies inherent in making and interpreting density measurements at the light microscopic level. Therefore, the terminal field volumes were calculated when any label, regardless of density, was present. This provides data on the topographical organization of the terminal fields but does not provide information concerning the absolute amount of afferent input into the NTS.
Rats were sedated with a 0.32 mg/kg injection of Domitor (medetomidine hydrochloride: Pfizer Animal Health, Exton, PA; I.M.) and anesthetized with 40 mg/kg Ketaset (ketamine hydrochloride: Fort Dodge Animal Health, Fort Dodge, IA; I.M.). A water-circulating heating pad was used to maintain body temperature. All rats except for those aged P15–P16 were positioned in a nontraumatic head holder. A ventral approach was taken in all rats to expose the CT and GSP nerves within the right tympanic bulla. The CT and GSP nerves were cut near the geniculate ganglion in the tympanic bulla and dimethyl sulfoxide (DMSO; Fisher Scientific Company, Fair Lawn, NJ) was briefly applied to the cut nerves. Crystals of 3-kD biotinylated dextran amine were then applied to the proximal cut end of the GSP and 3 kD Texas red dextran amine was applied to the proximal cut end of the CT (Fig. 1). A mixture of Vaseline and mineral oil was applied to prevent migration of dye. The IXth nerve was isolated just medial to the tympanic bulla and was cut and placed on a small piece of parafilm. Again, DMSO was applied briefly (~60 seconds) and crystals of 3-kD Cascade blue dextran amine were applied to the proximal cut end of the nerve (Fig. 1). All dextran amine conjugates were purchased from Invitrogen (Carlsbad, CA). The Vaseline and mineral oil mixture and a layer of parafilm were placed on top of the IXth nerve to keep the dye in place. Animals were then injected with 5 mg/kg Antisedan (atipamezole hydrochloride: Pfizer Animal Health; I.M.) to promote reversal of anesthesia. After an 18–24-hour survival, animals were deeply anesthetized with 4 mg/kg urethane (ethyl carbamate: Sigma-Aldrich Co., St. Louis, MO; I.P.) and transcardially perfused with Krebs-Henseleit buffer (pH 7.3), followed by 8% paraformaldehyde in PBS (pH 7.2).
We established previously (May and Hill, 2006 (link)) that 1) tracers placed on a nerve did not inadvertently label other nerves, 2) the full complement of fibers were labeled as revealed by examinations of the respective ganglia, and 3) the period of survival postsurgery was optimal for transport of each anterograde tracer. It should be noted that the mere presence of labeled terminal fields from GSP, CT, or IX axons does not necessarily mean that they convey taste information. For example, the three nerves also supply temperature and tactile information (Frank, 1968 ; Ogawa et al., 1968 (link); Smith et al., 1988 (link); Sollars and Hill, 1998 (link)). Thus, the NTS is a highly integrative center for multiple sensory inputs.