The Effects of Nicotine Receptor Blockers on Trigeminal Nerve Responses to Nicotine

Hessamadin Alimohammadi
Department of Biology
Wake Forest University
Winston-Salem, NC 27109
Introduction:

Trigeminal nerve fibers in the nasal cavity respond to a wide variety of chemical stimuli. These fibers are part of what has been called the common chemical sense. More recently, the term chemesthesis has been used to denote that trigeminal chemoreceptors are actually temperature and pain fibers, and therefore a part of the somatosensory system.

Trigeminal free nerve endings in the nasal cavity, arising from Ad and C fibers of the nasopalatine and ethmoid branches of the trigeminal nerve are scattered throughout the respiratory epithelium1,4. Although we know that chemical stimuli elicit responses from trigeminal nerve fibers, the mechanism of stimulation is not understood. Several lines of evidence suggest that the chemosensitivity of trigeminal nerve endings in the nasal cavity may be receptor mediated5,6. Evidence for nicotinic acetylcholine receptors (nAChRs) in trigeminal sensory neurons has come from experiments utilizing patch-clamps and biochemical techniques2,3. None of these studies have been able to demonstrate the existence of nAChRs on trigeminal nerve endings found in the epithelium of the nasal cavity. In the present study, we examined the possibility that trigeminal nerve responses to chemicals may be receptor-mediated. We tested the effects of the nicotinic receptor blockers mecamylamine hydrochloride and dihydro-b-erythroidine hydrobromide (DHBE) on trigeminal nerve responses to nicotine and cyclohexanone.


Methods:

A total of forty male Sprague-Dawley rats weighing between 350 and 750 grams were used. Each rat was anesthetized with urethane (ethyl carbamate: 1.0 g/kg injected i.p.). Two cannulae were inserted into the trachea of each rat. One cannula, open to room air, was inserted towards the rat’s lungs. A second cannula connected to a vacuum line was inserted rostrally into the nasopharynx to control the flow of air/stimuli through the nasal cavity. Each rat was then restrained in a head holder and the ethmoid nerve was exposed as previously described by Silver (1990). The exposed nerve was freed from neighboring tissue, cut several millimeters distal to the foramen, and stripped of its connective sheath. The nerve bundle was then placed on a pair of platinum-iridium wire hook electrodes, and the pocket of tissue surrounding the nerve was filled with halocarbon oil.

Odorants were presented using a computer-controlled air-dilution olfactometer8,10. Each rat was stimulated with either (-)-nicotine or cyclohexanone (12.5 ppm and 450 ppm, respectively). These odorants, at the concentrations used, have previously been shown to stimulate the ethmoid nerve7,9. Each rat received a total of 35 stimulus presentations, each lasting for 10 seconds, with 300 seconds between each presentation. Rats in experimental groups were treated with either dihydro-b-erythroidine hydrobromide or mecamylamine hydrochloride (2.5 X 10-5 mol/kg injected i.p.). Rats in the saline group were treated with physiological saline (1.0 ml/kg injected i.p.). All injections were made immediately after the first stimulus presentation.

Multiunit neural activity (Neural Response) from the ethmoid nerve was amplified and summated (Integrated Response) using an averaging circuit. Respiration was recorded by a thermocouple inserted into the rat’s breathing tube. Neural response, integrated response, and respiration were recorded for a period of 40 seconds for each stimulus presentation (12 seconds before, 10 seconds during, and 18 seconds after each presentation). The data were analyzed by multiple analyses of variance. Significance was examined using Tukey’s comparison of means at p < 0.05.


Results:
Figure 1. Experimental Set up
Figure 1.  Experimental setup. Odorants were presented via a computer-controlled air-dilution olfactometer.  Data were recorded using an automated acquisition system which was controlled by the olfactometer.  Note that respiration was monitored by a thermocouple inserted into the rat’s breathing tube. 
Figure 2. Sample record

Figure 2. Sample record blown up
Figure 2. Sample record. Neural response, integrated response, and respiration were recorded in arbitrary voltage units for a period of 40 seconds per stimulus presentation. The stimulus marker deflects down when the olfactometer is delivering an odorant. The integrated response curves were used to generate all data.  The magnitude of each response was measured as the difference in response magnitude between the onset and completion of stimulus presentation.
Figure 3a. Nicotine responses with and without blockers

Figure 3b. Structure of nicotine and response
 
 Figure 3. (a)  Changes in the magnitude of response to nicotine over a
 170 minute period (35 stimulus presentations).  Response magnitude
 values were converted to percentage values, calculated as percent of the
 first response.  (*) indicates that there was a significant difference
 between the experimental groups and the control and saline groups.  (+)
 indicates that the experimental groups were significantly different than
 the non-injected group (control).   (b)  Chemical structure of nicotine and
 a representative integrated response curve (smoothed for clarity).
 Figure 4a. Cyclohexanone responses with and without blockers
Figure 4b. Structure of cyclohexanone and response
Figure 4. (a)  Changes in the magnitude of response to cyclohexanone over a 170 minute period (35 stimulus presentations).  Response magnitude values were converted to percentage values, calculated as percent of the first response.  There were no significant differences between the experimental groups and the control group.  (b)  Chemical structure of cyclohexanone and a representative integrated response curve (smoothed for clarity).
Figure 5 Structures of blockers and ACh channels
Figure 5. (a)  Chemical structures of the nicotinic receptor blockers used in this study. (b)  Structure of a nicotinic acetylcholine receptor (nAChR).  Each nAChR is comprised of five subunits.  Only a subunits are involved in binding of ligand (modified from Kandel, 1991).


Conclusions: Together, these data imply that chemosensitive trigeminal nerve endings in the nasal cavity may contain a wide variety of mechanisms for the detection of chemical stimuli.

References:

1. Finger, T. E., M. L. Getchell, T. V. Getchell & J. C. Kinnamon. 1990. Affector and effector
        functions of peptidergic innervation of the nasal cavity. In: Chemical Senses. Vol. 2:
        Irritation. B. G. Green, J. R. Mason & M. R. Kare, eds. Marcell Dekker, New York. pp. 1-20.

2. Flores, C.M., DeCamp, R. M., Kilo, S., Rogers, S.W., & Hargreaves, K.M. 1996. Neuronal
        nicotinic receptor expression in sensory neurons of the rat trigeminal ganglion:
        demonstration of alpha3 beta4, a novel subtype in the mammalian nervous system. J.
        Neurosci. 16: 7892-7901

3. Liu, L., Pugh, W., Ma, H., & Simon, S. A. 1993. Identification of acetylcholine receptors in
        adult rat trigeminal ganglion neurons. Brain Research. 617: 37-42.

4. Lundblad, L., J. M. Lundberg, E. Brodin & A. Änggård. 1983. Origin and distribution of
        capsaicin-sensitive substance P-immunoreactive nerves in the nasal mucosa. Acta
        Otolaryngol. 96: 485-493.

5. Nielsen, G. D. 1991. Mechanisms of activation of the sensory irritant receptor by air-borne
        chemicals. CRC Crit. Rev. Toxicol. 21: 183-208.

6. Nielsen, G. D. & Y. Alarie. 1982. Sensory irritation, pulmonary irritation, and respiratory
        stimulation by airborne benzene and alkylbenzenes: prediction of safe industrial exposure
        levels and correlation with their thermodynamic properties. Toxicol.Appl. Pharmacol. 65:
        459-477.

7. Silver, W. L. & D. G. Moulton. 1982. Chemosensitivity of rat nasal trigeminal receptors.
        Physiol. Behav. 28: 927-931.

8. Silver, W. L., D. B. Walker, M. W. Ogden & J. C. Walker. 1990. Nasal trigeminal
        responses to toluene presented by an automated delivery system. Chem. Senses. 15:
        701-712.

9. Walker, J. C., M. Kendal-Reed, C. J. Keiger, M. Bencherif & W. L. Silver. 1996. Olfactory and
        trigeminal responses to nicotine. Drug Dev. Res. 38: 160-168.

10. Youngentob, S. L., M. M. Mozell, P. R. Sheehe, D. E. Hornung. 1987. A quantitative analysis
        of sniffing strategies in rats performing odor detection tasks. Physiol. Behav. 41: 59-69.