ARTIFICIAL SWEETENERS, NaCl, AND KCl STIMULATE PERIPHERAL TRIGEMINAL NERVE CHEMORECEPTORS Vajini N. Atukorale, Matthew W. Greene, and Wayne L. Silver Department of Biology, Wake Forest University, Winston-Salem, NC Methods Rats were anesthetized with urethane (ethyl carbamate: 1 g/kg injected i.p.). Two cannulae were inserted into the trachea of each rat. One cannula allowed the rat to breathe room air. The second cannula, inserted into the nasopharynx, was connected via a pump to a reservoir containing Ringer’s solution. Stimuli (1.0 ml) were injected into the flow of Ringer’s (10 ml/min), which was allowed to drip from the rat’s nose. Rats were restrained in a head holder and the ethmoid nerve was exposed and placed on a pair of platinum-iridium wire hook electrodes. Multiunit neural activity from the ethmoid nerve was summated using an averaging circuit. The data were analyzed by taking the maximum height of the integrated response after stimulus delivery. Responses are reported as a percentage of the standard response to 10µM capsaicin. Concentrations are reported for the injected solutions. Introduction The trigeminal nerve provides sensory information from the eyes, nose, and mouth. It is a multisensory nerve, responding to a variety of irritants in the environment. Several receptor proteins associated with the trigeminal nerve mediate these responses. Artificial sweeteners, which are of obvious economic importance, are reported to be irritating at high concentrations. As irritants they should stimulate the trigeminal nerve. The present study examined peripheral trigeminal nerve responses to increasing concentrations of three artificial sweeteners (Sodium Saccharin, Sodium Cyclamate, and Acesulfame K), NaCl, and KCl. Literature Cited Finger TE, Böttger B, Hansen A, Anderson KT, Alimohammadi H, and Silver WL. (2003) Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration. PNAS. 100: Dinh, Q.T., Groneberg, D.A., Mingomataj, E., Peiser, C., Heppt, W., Dinh, S., Arck, P.C., Klapp, B.F., and Fischer, A. (2003) Expression of substance P and vanilloid receptor (VR1) in trigeminal sensory neurons projecting to the mouse nasal mucosa. Neuropeptides 37, Kuhn C, Bufe B, Winnig M, Hofmann T, Frank O, Behrens M, Lewtschenko T, Slack JP, Ward CD, Meyerhof W. (2004). Bitter taste receptors for Saccharin and Acesulfame K. J Neurosci. 24: Riera C, Damak S, Coutre JL. (2006). Food flavors and the sweetener Saccharin activate the transient receptor potential vanilloid subtype 1 (TRPV1) channel. Chem Senses. 31: A49-A50. Conclusions The artificial sweeteners tested, Sodium Saccharin, Sodium Cyclamate, and Acesulfame K, elicit graded responses (with respect to increasing concentrations) from the ethmoid branch of the trigeminal nerve. Artificial sweeteners stimulate TRPV1 receptors (capsaicin, irritant receptors) (Riera et al., 2006) and T2R receptors (bitter receptors) (Kuhn et al., 2004) in vitro. As both of these receptor proteins are associated with trigeminal nerves (Dinh et al., 2003; Finger et al., 2003), either or both may contribute to the trigeminal nerve responses to the sweeteners tested. NaCl and KCl also elicited responses from the trigeminal nerve. Therefore, Na + and K + may have played a role in the response to the sweeteners tested. However, the thresholds for NaCl and KCl were higher than those of the sweeteners, suggesting that the sweeteners must be stimulatory by themselves. Future experiments will examine trigeminal nerve responses to the sweeteners in the presence of TRPV1 and T2R receptor blockers. Figure 1. Electrophysiological setup. Stimuli (1.0 ml) were delivered via a syringe into Ringer’s solution flowing through the rat’s nose. Responses were digitized and recorded on an IBM computer with Acknowledge software (Biopac). Figure 2. Stimuli tested. Figure 1. Experimental Setup Amplifier Electrodes Audio Monitor Integrator Data Acquisition System Pump ← ← ↓ Nasopharyngeal tube Stimulus injection port Rat Ringer’s NaCl KCl Figure 2. Stimuli Tested Capsaicin 10 µM Capsaicin Acesulfame K 10 sec Sodium Cyclamate Sodium Saccharin 10mM100mM 250mM1000mM 750mM500mM NaCl KCl Figure 3. Integrated Responses Concentration (mM) % Capsaicin Response NaCl Sodium Saccharin Sodium Cyclamate Acesulfame K KCl Figure 4. Concentration-Responses Curves Figure 3. Multiunit, integrated responses to the stimuli tested at increasing concentrations. Figure 4. Concentration-response curves for the stimuli tested. Responses are reported as a percent of the response to 10 µM capsaicin. Error bars have been omitted for clarity. N=4 Acesulfame-K (potassium salt) Sodium Saccharin Sodium Cyclamate