James C. Walker

Individual variation in the expression profiles of nicotinic receptors in the olfactory bulb and trigeminal ganglion and identification of α2, α6, α9, and β3 transcripts

Abstract Nicotine evokes dose-dependent and often variable chemosensory responses in animals and humans. Earlier observations that nicotine binds to some nicotinic acetylcholine receptor (nAChR) subtypes in the olfactory bulb (OB) and trigeminal ganglion (TG) led us to investigate the complete nAChR expression profile in each tissue and to determine whether inter-individual differences exist in male and female rats. Total RNA was extracted from individual samples of dissected OB and TG and analyzed by a sensitive reverse transcription–polymerase chain reaction (RT–PCR) assay to determine the messenger RNA profiles of ten transcripts encoded by the α2, α3, α4, α5, α6, α7, α9, β2, β3, and β4 nAChR genes. We found that (a) in the OB, all animals expressed α2, α3, α4, α5, α7, β2, and β4 subunit mRNAs, whereas α6, β3, and α9 transcripts were expressed in only 17, 28, and 33% of the animals, respectively, and (b) in the TG, all animals expressed α2, α3, α6, α7, β2, and β4 subunit mRNAs, whereas α9, β3, α4, and α5 transcripts were expressed in 4, 38, 88, and 92% of the animals, respectively. These results also identified new subunits that are expressed in each tissue (α2, α6, α9, and β3) and demonstrated that individual rats may have different tissue-specific expression profiles for α4, α5, α6, α9, and β3 transcripts. Such variations are likely to be reflected in the composition of functional receptor subtypes in the rat OB and TG that have different activation and desensitization characteristics to acetylcholine and nicotine.

Olfactory and trigeminal responses to nicotine

Olfactory and trigeminal sensitivities to vapor‐phase nicotine were assessed by using psychophysical studies with normal and anosmic human subjects and using electrophysiological studies with rats and pigeons. This work showed that 1) psychophysical estimates of sensitivity are approximately tenfold higher (i.e., lower thresholds) than those based on neural recordings, with both techniques demonstrating greater olfactory than trigeminal sensitivity for nicotine and other compounds; 2) for both chemosensory inputs, sensitivity to nicotine is at least 30‐fold greater than that to several other compounds; 3) human subjects can discriminate qualitatively between the S‐(−) and the R‐(−) stereoisomers of nicotine, although the relative importance of olfactory and trigeminal inputs in this discriminative ability is unclear; and 4) trigeminal nerve responses in rats show similar thresholds for S‐(−)‐ and R‐(+)‐nicotine but show lower suprathreshold responses to the R‐(+) stereoisomer. The olfactory epithelium and trigeminal ganglion exhibit high‐affinity binding of S‐(−)‐nicotine. In addition, reverse transcriptase‐polymerase chain reaction (RT‐PCR) studies have shown that many of the nicotinic acetylcholine receptor (nAChR) subunits found in other parts of the nervous system are present in the olfactory epithelium and bulb and in the trigeminal ganglion. Collectively, these findings suggest that two or more of the types of nAChRs identified in other parts of the nervous system may serve as receptor proteins that bind nicotine‐like odorants or irritants. Investigation of the pharmacology of chemosensory responses to nicotine may help to establish causal links between specific receptor proteins and the perception of odor and irritation. Drug Dev. Res. 38:160–168

Comparison of Odor Perception in Humans and Animals

In this Chapter the abilities of humans and animals to detect odorants and to perceive the strengths of suprathreshold concentrations of odorants are compared and an effort is made to provide conceptual and practical reasons for such comparisons. In addition, current obstacles to more profitable use of psychophysical data for determining the neural mechanisms that underlie human odor perception are discussed. Results from odor psychophysical experiments in humans and animals are then summarized and general conclusions and recommendations for future research are offered.

Assessing the effects of odorants on nasal airway size and breathing.

A technique was developed to obtain continuous measurements of both respiratory behavior and nasal patency in response to well-controlled odorant stimulation. An automated apparatus similar to that described by Walker et al. (27) was used to present precise concentrations of an odorant. The pressure-flow technique (28) was used to continuously measure nasal airway cross-sectional area, nasal airflow rate, air volume and time characteristics associated with breathing before and during odorant stimulation. Immediately following each odorant presentation, subjects entered their psychophysical responses into a microcomputer via an electronic mouse. Respiratory and psychophysical responses of ten normal subjects to four concentrations of acetic acid during eight odorant trials were recorded; eight clean-air trials were also conducted. At the highest concentration, changes in respiratory behavior were observed as early as 200 ms after stimulus onset in some subjects. Inspiratory volumes during odorant presentation were lower than those seen just before stimulation. The magnitude of this decrease was directly related to the concentration of acetic acid and to the perceived intensity of the odor and degree of nasal irritation.

Nicotinic Cholinergic Receptor Expression in the Human Nasal Mucosa

Twenty-four nasal mucosa specimens were obtained from the inferior or middle turbinates of 6 normal subjects and 18 patients with chronic sinusitis, inflammatory polyp formation, or sinus allergies. Reverse transcription—polymerase chain reaction analysis was used to identify the non-neuronal nicotinic cholinergic receptor (nAChR) subunits that were expressed in the nasal mucosa. Collectively, transcripts for α (α1, α2, α3, α4, α6, α7) and β (β2, β3, β4) nAChR subunit genes were detected in the respiratory mucosa. The α3, α7, and β2 subunits were expressed in 92%, 88%, and 75% of the subjects, respectively. There was a high degree of interindividual variation in nAChR subunit gene expression among subjects. A significant univariate association was found between tissue type and β4 expression and between gender and β3 expression. These data suggest that cells in the nasal mucosa express the necessary messenger RNAs (mRNAs) for numerous nAChR combinations. Moreover, our identification of nAChR subunit mRNAs in the nasal mucosa extends the findings of other functional studies of nAChRs in nasal epithelial cells and implies that nicotine from tobacco products such as cigarette smoke and nicotine nasal spray may have direct cellular effects on nasal mucosa cells through activation of homogeneous or heterogeneous nAChRs. A significant number of patients receiving nicotine nasal spray have reported nasal irritation, and there are reports of transient irritation of the throat and trachea with the use of smoke-free nicotine cigarettes. These adverse respiratory effects may be due to activation of nAChRs in epithelial cells of the nose and trachea.

Time course of reinnervation of the olfactory bulb after transection of the primary olfactory nerve in the pigeon.

Horseradish peroxidase (HRP) histochemistry was used to study the time course of reinnervation of the pigeon olfactory bulb after simple transection of the primary olfactory nerve. At selected intervals (9, 13, 29, 61 and 93 days) after transection of the right olfactory nerve, a concentrated solution of HRP was instilled in both nasal cavities. Intracarotid perfusion was performed 3 days after nasal instillation of HRP and 40-microns sections of olfactory bulb processed with the tetramethylbenzidine (TMB)-HRP histochemical protocol to visualize olfactory receptor axon terminals reinnervating the glomerular layer of the bulb. The total area of reinnervation of four representative regions in the bulb of the transected side were compared with that on the control bulb. The area of innervation by newly reconstituted olfactory axons was approximately 17% of control values at the 12-day posttransection time interval. A progressive increase in the area of reinnervation was observed over time. Reinnervation of the right bulb was approximately 70% complete at the 32-day posttransection time interval and indistinguishable from the left control bulb at the 64- and 96-day posttransection time intervals. A uniform pattern of reinnervation of different bulb regions was observed at all time intervals. These results indicate that the peripheral olfactory system of the pigeon is capable of complete reconstitution after nerve transection. Our findings should be useful in guiding functional comparisons of normal and newly reconstituted peripheral olfactory systems in the pigeon.

Method for calibrating olfactometer output. Part 1. Acetic and propionic acids

An analytical method for measuring acetic acid and propionic acid in air was developed and validated. This method entails collection of acetic acid and propionic acid on XAD-7 sorbent tubes connected to an olfactometer and extraction of the sorbent in a solution of methanol and formic acid. The acids are then determined by gas chromatography with flame ionization detection. This methodology was successfully implemented in calibration of olfactometer output.