Physiology of Vertebrate Olfactory Chemoreception

1 Physiology of Vertebrate Olfactory

Chemoreception

THOMAS V. GETCHELL and M A R I L Y N L. GETCHELL

Introduction

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I. A n a t o m i c a l and Cellular Basis o f Olfactory Function

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I I . N e u r o p h y s i o l o g y o f Olfactory Reception

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A . M e t h o d o l o g y and T e r m i n o l o g y

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B. Cellular Mechanisms Resulting from O d o r A c t i v a t i o n .

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C. Peripheral Mechanisms o f Discrimination and C o d i n g

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I I I . M o l e c u l a r B i o l o g y o f O d o r Detection

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I V . S u m m a r y : Schema Outlining the Sequence o f Events L e a d i n g to the Transmission o f Olfactory Information to the Brain

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References

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INTRODUCTION

This chapter begins with a brief summary of the neuroanatomical organiza­ tion of the peripheral olfactory system, covering primarily the olfactory epithelium, with lesser emphasis on the olfactory bulb and higher centers in the olfactory pathway. The major focus is a detailed discussion of the physiological activity of olfactory receptor neurons as shown primarily by electrophysiological techniques, and the interaction of odorants with the chemoreceptive membrane of these cells. The results of psychological and biochemical experiments are integrated with those from neurophysiological studies, insofar as they are related to considerations of coding of odor quality and adaptive, transductive, and molecular recognition mechanisms. A

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CHEMISTRY

1982 by A c a d e m i c Press, I n c . ,

A l l rights o f r e p r o d u c t i o n in any f o r m reserved.

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Thomas V . Getchell and Marilyn L . Getchell

summary of perireceptor and olfactory receptor events initiated by the nasal inhalation of an odor concludes the chapter. Detailed neurophysiological correlates related to olfactory discrimination in higher cerebral centers are beyond the scope of the chapter, as is behavior. This communication draws on relevant literature through the summer of 1978. Recently, several excellent books, symposium volumes, and review articles have been published that offer a broader spectrum of topics related to vertebrate olfactory chemoreception than those treated in this chapter. They include: Methods of Olfactory Research, Moulton, Turk, and Johnston (eds.) (1975); Chemical Signals in Vertebrates, Muller-Schwarze and Mozell (eds.) (1977); Mammalian Olfaction, Reproductive Processes, and Behavior, Doty (ed.) (1976); Food Intake and Chemical Senses, Katsuki, Sato, Takagi andOomura(eds.) (1977); Olfaction and Taste VI, Le Magnen and MacLeod (eds.) (1977); Neurochemistry of Olfactory Circuits, in Society for Neuroscience Symposia Vol. III, Ferrendelli (ed.); The olfactory system: A model for the study of neurogenesis and axon regeneration in mammals, Graziadei and Monti-Graziadei, in Neuronal Plasticity, Cotman (ed.); Biochemical Markers of the Primary Olfactory Pathway, Margolis, in Advances in Neurochemistry, Agranoff and Aprision (eds.); Physiology of Olfactory Reception, Gesteland, in Frog Neurobiology, Llinâs and Precht (eds.); Structure-Activity Relationships, G. Benz (ed.) (1976); and Transduction et codage des informations olfactives chez les vertèbres, Holley and MacLeod.

I. ANATOMICAL AND CELLULAR BASIS OF OLFACTORY FUNCTION

The sensory cells that detect volatile, low-molecular-weight organic molecules (stimulus, odor, odorant, olfactant) are olfactory receptor neurons. In humans they are located in a relatively small area (about 2.5 cm ) of each nasal cavity called the olfactory sensory epithelium (Fig. 1). This epithelium covers a portion of the lateral wall of the medial nasal septum and the medial wall of the superior concha turbinai. Air bearing the volatile molecules enters the nasal cavity through the external naris and is carried over the surface of the epithelium with each inspiration during normal breathing. The air then passes through the internal naris toward the lungs. With each expiration the reverse process occurs. Therefore, air currents initiated by the rhythmic contractions of muscles of the diaphragm and the thoracic wall during the normal breathing cycle are initially responsible for exposure of the olfactory epithelium to molecules that may evoke odor sensations. The olfactory epithelium has a remarkably similar cellular organization throughout higher animal species (Graziadei, 1971). It is approximately 2

3

1. Olfactory Neurophysiology

Cribriform plate iFila olfactoria

uJSft^ Medial nasal septum Turbinais

External naris L- Internal naris Fig. 1.

D r a w i n g o f the bisected human nasal cavity showing the general organization,

macroscopic anatomical components, and passageway for air bearing o d o r molecules. ( M o d i f i e d from R. J. Christman, Sensory Experience,

Intext Educational Publishers, 1971.)

150-300 μιη thick and contains three primary cell types—olfactory receptor neurons (ORC), sustentacular cells (SC), and basal cells (BC)—with their nuclei forming three distinct strata [Fig. 2(a)]. A layer of mucus ( M U ) about 10-50 μιη thick, through which the molecules travel prior to their interaction with the cellular elements, continually bathes the epithelial surface. It sweeps over the surface at rates estimated to range from 10 to 60 mm/min (Moulton and Beidler, 1967). Approximately 50 χ 10 olfactory receptor neurons com­ prise the sensory epithelium on each side of the nasal cavity, there being about 10 receptor neurons/100 μιη . An olfactory receptor cell is the prototype of a bipolar neuron [Fig. 2(a)]. From the soma, which lies in the intermediate nuclear stratum [ORC, Fig. 2(b)], a dendrite approximately 1-2 μιη in diameter extends distally to the epithelial surface. There it terminates in a knob from which several long cilia project into the mucus. The cilia are up to 200 μιη in length. Proximally, the receptor cell axon projects into the submucosa (SM), where it joins with other axons to form the fila olfactoria of the olfactory nerve. The fila—LG., bundles of olfactory receptor cell axons surrounded by Schwann cell (glia) and connective tissue elements—project through the cribriform plate of the ethmoid bone to enter the cranial cavity (Fig. 1). The axons course to the olfactory bulb where they synapse with the second-order neurons, the mitral/tufted cells. The unmyelinated axons of the receptor neurons are among the smallest in the body, with a modal crosssectional diameter of 0.2 μιη. They project to the olfactory bulb without branching or making synaptic contacts with other neurons. Low-resistance channels (gap junctions) to provide lateral connectivity between adjacent 6

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Thomas V . Getchell and Marilyn L . Getchell Q

Fig.

2.

Epithelium

Photomicrographs

b

Receptor

cell

(a, d right) and drawings ( b , c, d left) showing the cellular

organization o f the olfactory epithelium ( a ) and its major cellular components ( b , c, d ) . F o r full description, see text. ( F r o m Getchell and Getchell, 1977b.)

receptor neurons are few or absent. Efferent (motor) input from the brain to the receptor neuron is also lacking. Because of the lack of efferent feedback, electrotonic coupling, or synaptic interactions between receptor neurons, it is generally considered that the first-order receptor neurons function as independent physiological units. A second type of cell in the epithelium is the sustentacular cell [Fig. 2(c)]. It typically extends the full thickness of the olfactory epithelium. Super­ ficially, sustentacular cells envelop the receptor cell dendrites and serve to isolate them from one another by forming a hexagonal array when viewed from the epithelial surface. The apical portions of the receptor cell dendrite and sustentacular cell are bound together by tight junctions at the mucosal surface. These presumably serve three functions: first, mechanical, to main­ tain the structural integrity of the distal epithelial surface ; second, isolation,

1. Olfactory Neurophysiology

5

to separate the overlying mucus from the extracellular fluid compartment surrounding the cells; and third, diffusion barrier, to impede migration of primarily non lipid-soluble molecules through the epithelium. The apical regions of adjacent sustentacular cells also bear gap junctions that provide a network of lateral connectivity among sustentacular cells. Sustentacular cell nuclei form the distal nuclear layer (SC) in the olfactory epithelium [Fig. 2(a)]. The subnuclear stalk projects proximally and ramifies into the foot processes, which appear to be splayed over the basement membrane. The supranuclear region extends to the epithelial surface, where numerous short (less than 10 μιη) microvilli extend into the mucus. This region also contains vesicle-associated material that, upon histochemical staining, was identified as acidic and possibly sulfated mucopolysaccharides (Getchell and Getchell, 1977b). The irregularly shaped basal cells, with their nuclei forming the most proximal nuclear stratum (BC), are found deep in the olfactory epithelium [Fig. 2(a)]. Although the precise role that these cells perform in the integrated epithelial function remains elusive and controversial, considerable morpho­ logical (Graziadei and Monti-Graziadei, 1978) and biochemical (Margolis, 1975, 1977) evidence suggests two functions. First, progenitor cells are cyclically removed from this population to develop into functionally mature olfactory receptor neurons during normal cell turnover. Second, subsequent to traumatic chemical insult where the olfactory epithelium may become necrotic with concomitant loss of olfactory sensory function, certain cells in the basal region serve as the stem-cell population for regeneration of the sensory epithelium with return of olfactory function. Large multicellular Bowman's glands lie primarily in the submucosa [Fig. 2(a) and (d)]. They are exocrine glands which open to the epithelial surface via secretory ducts that traverse the olfactory epithelium. The secretory cells that comprise the acinus contain granule-associated material, identified as neutral mucopolysaccharides upon histochemical staining (Getchell and Getchell, 1977b). In summary, the olfactory epithelium consists of three principal cell types: olfactory receptor, sustentacular, and basal. The main function of the receptor neurons is to detect, encode, and transmit information about the intensity, duration, and quality of the odorant to the olfactory bulb and higher cortical centers (for review, see MacLeod, 1971; Shepherd, 1972; Shepherd et al, 1975). The basal cells appear to be stem cells that become developmentally active during normal cell turn-over and rejuvenation of the olfactory epithelium. The sustentacular cells and acinar cells of Bowman's gland appear to contribute specific types of mucopolysaccharides as secretory products to the mucus layer covering the epithelial surface.

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Thomas V . Getchell and Marilyn L . Getchell

IL NEUROPHYSIOLOGY OF OLFACTORY RECEPTION

Electrophysiological investigations have provided two basic types of information about the functioning of olfactory receptor neurons. The first is determination of the sequence of neural events resulting from the molecular interaction of odors with the chemoceptive membrane leading to the trans­ mission of electrical signals to the brain. The second is related to questions of molecular discrimination, odor intensity, and quality coding. Each of these topics is discussed sequentially after a brief introduction to the methodology and terminology. A . Methodology and Terminology

When olfactory stimuli are delivered to the olfactory epithelium, three types of voltage transients may be recorded from the epithelium using appropriate electrophysiological recording techniques. The first type (Fig. 3, left) is recorded as a slow voltage change from the epithelial surface and was called the electroolfactogram by Ottoson (1956). Although it is typically

Fig. 3.

A collage showing the relationship between the cellular organization o f the olfactory

epithelium and the positions o f the extracellular recording electrodes e m p l o y e d to monitor the population response, V e o g (left), and unitary spike potentials from single olfactory receptor neurons (right) in response to o d o r stimulation. O n the right side o f the figure, Β shows the back­ ground spontaneous activity recorded from 2 receptor neurons as represented by the large and small amplitude spike potentials. T h e time base is expanded in A to show the triphasic ( + , — , + ) voltage conformation o f the large amplitude spike. T h e trace shown in C represents an excitatory discharge in response to o d o r stimulation. T h e receptor neuron represented by the smaller amplitude spike was nonresponsive to the o d o r . ( F r o m Getchell and Getchell, 1974.)

1. Olfactory Neurophysiology

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monophasic negative, it may be composed of two voltage components in which the large amplitude negative component is preceded by an initial positive voltage transient. For example (Fig. 3, left), rc-butanol evokes a biphasic voltage transient which has a duration of about 4 s with a smallamplitude positive transient, Veog( + ), followed by the larger amplitude negative voltage component, Veog( — ) . In general, Veog( — ) is considered to be a summated receptor potential: that is, it represents an excitatory response generated by many receptor cells in response to odor stimulation. The amplitude of Veog( — ) increases systematically with odor concentration (Ottoson, 1956, 1971; Getchell, 1974b). This indicates that the responsive receptor neurons are becoming more depolarized and that less-sensitive receptor neurons are being activated with increasing odor concentration. Several lines of evidence support these conclusions regarding the significance of Veog( - ) (e.g., Ottoson, 1970,1971 ; Getchell; 1974b; Getchell and Getchell, 1977a). Three separate mechanisms have been proposed in efforts to account for V e o g ( + ) . They are: first, an electrochemical recording artifact (Ottoson, 1956; Muller, 1971); second, an inhibitory response generated by olfactory receptors in response to odor stimulation (Gesteland, 1964,1967); and third, a secretory response generated by sustentacular cells in response to odor stimulation (Okano and Takagi, 1974). Detailed investigation of the response properties of Veog( + ) showed that it has a cellular source, and that a secretory process is consistent with the interpretation of Veog( + ) (Getchell, 1974b, 1977a,b). The second type of voltage transient is a unitary action potential recorded extracellularly from an olfactory receptor neuron during an intraepithelial electrode excursion (Fig. 3, right). The individual action potential (spike, impulse) is generally of low amplitude and of brief duration, i.e., less than 8 ms (A). Many receptor neurons exhibit spontaneous activity, that is, impulse discharges in the absence of experimentally introduced stimuli (B). The rate varies over a wide range from 0.07 to 1.8 spikes/s, with most receptor cells (68 % ) having a very low rate of less than 0.4 spikes/s (Getchell, 1974a). This is a very low rate of spontaneous activity compared with activity of other neurons, e.g., about 46 spikes/s for auditory nerve fibers. There are divergent views concerning the role that spontaneous activity may play in the transmission of sensory information. One is that it simply reflects biological noise. The other is that it serves as a dynamically set reference point that may have significance in the detection of near-threshold chemical stimuli (Getchell, 1974a; Getchell and Getchell, 1974; Van Drongelen, 1978). In response to odor stimulation, a responsive receptor neuron exhibits an excitatory discharge of impulses, with a characteristic sequence of interspike intervals (C). Increasing odor concentration results in a simple increase in discharge frequency.

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Thomas V . Getchell and Marilyn L . Getchell

The third type of voltage transient (Fig. 4) is a transmembrane voltage change that most clearly delineates the coupling mechanism between the molecular activation of the receptor neuron and subsequent electrical events. It is obtained by recording intracellular^ with extremelyfine-tippedmicropipettes (<0.1 μιη). Although obtaining such recordings is difficult when adequate electrophysiological controls and cell identification techniques are used, it does provide insight into transmembrane mechanisms associated with cellular activation by odors (Aoki and Takagi, 1968 ; Farbman and Gesteland, 1974; Getchell, 1977a,b; Suzuki, 1977). In summary, each type of electrophysiological recording technique provides different information about the activity of the cellular components of the olfactory epithelium: the first, Veog, population events; the second, α

Generator

mechanism

b

Spike

c

G1ial-type

depolarization

d

Secretory

Onset , V e o g ( - )

Fig. 4.

electrogenesis

process

Onset, Veog (-)

Schema proposed for the o d o r e v o k e d processes activated in olfactory receptor (a, b )

and sustentacular (c, d ) cells. F o r full description see text. ( F r o m Getchell, 1977b.)

1. Olfactory Neurophysiology

9

action potentials, single receptor cell events; and the third, intracellular^ recorded voltage transients, transmembrane events.

B. Cellular Mechanisms Resulting from Odor Activation

The interaction of certain molecules with the olfactory epithelium provides a wealth of information to the organism about the changing chemical environment. This information may elicit the perception of the chemical stimulus as an odor sensation. The underlying sequence of neural mecha­ nisms, operable in the olfactory epithelium, that initiates these subsequent responses has been analyzed using electrophysiological and neuroanatomical techniques as described above. The sequence is now briefly summarized. Olfactory stimuli traverse the layer of mucus prior to their interaction with molecular receptors located on the cell membranes of the distal region of the receptor cell, either on the cilia or apical knob. The resultant molecular interaction, coupled with specific transmembrane ion conductance mecha­ nisms, initiates generator current flow into the dendritic region of the receptor cell [Fig. 4(a)]. Although the precise location of receptor sites remains uncertain at this time, the results of step intraepithelial Veog( — ) recordings support the hypothesis that distal elements in the olfactory epithelium function as a current sink activated by odors (Ottoson, 1956; Byzov and Flerova, 1964; Getchell, 1977a). If particular receptor site types are randomly distributed and not spatially restricted to a single cilium or ciliary region, the olfactory knob is the first site of signal integration in the olfactory system. The generator current then spreads electrotonically through the dendrite into the receptor cell soma, where an intracellular micropipette records a transmembrane depolarization, the generator potential, upon which action potentials are typically superimposed [Fig. 4(a)]. The electrical properties of the receptor cell membrane (Suzuki, 1977) and the calculated specific internal resistance of the cytoplasm indicate that the length constant is sufficient to allow for the centripetal electrotonic spread of current to the site of spike electrogenesis in the soma-initial axonal segment (Ottoson and Shepherd, 1967; Getchell, 1977b). Excitatory responses evoked by odors are characterized by a systematic decrease in the durations of the first several interspike intervals. This characteristic sequence presumably reflects the initial rapid invasion of generator currents triggered by the olfactant to the site of spike electrogenesis (Getchell, 1974a). Generator current in the soma-initial axonal segment subsequently activates the regenerative spike electrogenic mechanism (Getchell, 1973). It also represents the second site of signal integration in the receptor neuron. Current from the regenerative process invades the dendritic and axonal regions of the receptor cell, where

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Thomas V . Getchell and Marilyn L . Getchell

action potentials of distinctive polarities are recorded extracellularly as is shown in Fig. 4(b). The resultant pattern of impulses transmitted by the receptor cell axon to the olfactory bulb results from a complex interplay of odors with the chemoreceptive membrane, specific transmembrane ion conductance changes, and flow of ionic current. These are associated with specific regions of the olfactory receptor neuron. Sustentacular cells perform an important role in maintaining the mucoid and extracellular fluid compartments of the olfactory epithelium. A secretory process [Fig. 4(d)] activated in direct response to odors has been proposed for sustentacular cells based on electron microscopic observations (Bloom, 1954; Graziadei, 1971; Okano and Takagi, 1974; Reese, 1965), electro­ physiological recordings from the epithelial surface (Okano and Takagi, 1974) and intracellular recordings (Getchell, 1977a,b). For example, Okano and Takagi (1974) reported histological evidence of secretory activity only when certain stimuli such as chloroform or rerf-butyl alcohol evoked Veog( + ). Odors that evoked Veog( — ) caused no apparent release of secretory products. This implies that certain types of compounds commonly identified as anaesthetics or irritants preferentially release secretory products from sustentacular cells. The release of these products, perhaps acidic mucopoly­ saccharides, may provide a crucial protective factor for the apical portion of the olfactory receptor neuron by changing the composition of mucus in close proximity to the epithelial surface. Intracellular recordings from sustentacular cells also show transmembrane depolarizations [Fig. 4(c)] similar to those recorded from identified glial cells surrounding optic nerve fibers and neurons in the central nervous system. The membrane depolarizations may result from the accumulation of potassium ions in the extracellular space between the olfactory receptor neurons and sustentacular cells. The increase is probably due to odor-evoked olfactory receptor cell activity. Therefore, sustentacular cells perform several important functions in the integrated activity of the olfactory epithelium. First, they maintain the structural integrity of the epithelium through tight junctions with the receptor neurons; second, they discharge secretory material into the mucus layer, and under certain conditions they contribute specific types of mucopolysaccharides to the olfactory mucus; and third, they are involved in the regulation of the ionic environment of the cellular elements of the olfactory epithelium. In summary, odors interact with molecular receptors on the chemoceptive membrane of the olfactory cilia and knob. Current flow in the distal parts of the cell spreads electrotonically to the initial axonal segment where spike electrogenesis occurs. In addition, olfactants may cause the release of secre­ tory products from sustentacular cells, changing the nature of the mucus bathing the epithelial surface. Sustentacular cells may also function as stabilizers of the ionic environment of the external milieu.

1. Olfactory Neurophysiology

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C. Peripheral Mechanisms of Discrimination and Coding

The sequence of olfactory receptor cell and perireceptor events that leads to the transmission of information to the brain about the changing chemical environment has been described. The informational content encoded in the discharge pattern of impulses recorded from the receptor cell conveys the essential parameters of the stimulus, that is, the concentration and duration of exposure of the receptor neuron to the olfactant. One realizes from introspective experience that the olfactory world of man is quite rich with a wide variety of odor sensations. The variety is not unlike the myriad colors detected by the visual system. It was anticipated that the functional organiza­ tion of the olfactory epithelium would be analogous to that of the retina, where there are only three types of retinal receptors, each one most sensitive to a different wavelength of light—i.e., red, 611 nm; green, 530 nm; and blue, 460 nm. It was initially predicted from perceptual studies seeking to define primary odor categories (e.g., Linnaeus, 1752; Moncrieff, 1949; Amoore, 1964; for reviews of these earlier works see Engen, 1971; Davies, 1971; Köster, 1975; Moncrieff, 1977; Amoore, 1977b) that individual olfactory receptor neurons would be most responsive to those compounds represented by one primary odor category. Hence, if 10 primary odor categories were constructed from perceptual information, then the neurophysiologist would expect to find 10 different types of olfactory receptor neurons, each type being responsive to all constituents of one primary odor category and unresponsive to constituents in the other nine categories. In numerous electrophysiological experiments from different laboratories, employing a variety of odors, this was found not to be the case (for reviews see Gesteland, 1971; Holley and MacLeod, 1977; also Revial et α/., 1978a). Rather, each olfactory receptor neuron can detect a variety of odors, which cuts across psychologically defined odor classifications. For example, if olfactants A and M evoke distinctly different odor sensations, then they would represent different primary odor categories. If olfactant A* resembled A more than M , then it would be placed in category A; likewise M * with M . Essentially, unit re­ cordings from olfactory receptor cells clearly indicate that individual receptor neurons are responsive to any one, any combination, or all odors represented by A, A*, M, and M*. The issue is further clouded by the obser­ vation that although A and A* may evoke extremely similar odor sensations, they may have distinctly different molecular configurations, functional groups, and/or physicochemical properties—e.g., cyclopentadecanone and ethylene brassylate ; nitrobenzene and benzaldehyde (for reviews of structureactivity relationships, see Beets, 1971; Amoore, 1977a; Polack, 1973). Due to differential responses of receptor neurons to a variety of different odors and the lack of harmony with perceptually constructed odor categories, it is

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Thomas V . Getchell and Marilyn L . Getchell

postulated that different types of molecular receptor sites are found on the chemoreceptive membrane of each receptor neuron. Hence, at this point, it is difficult to account for the coding of perceived odor quality by the peripheral olfactory system in simplistic terms. The reader is directed to several review articles on this fascinating aspect of the problem (Gesteland, 1971, 1976; Beets, 1971; Holley and MacLeod, 1977). The results of electrophysiological experiments recording from olfactory receptor neurons should not be construed to mean that categories of primary odors do not exist. Rather, adequate criteria may not have been employed to construct categories of perceptually defined odor primaries; olfactory receptor neurons may order categories of odors using different criteria than those used in perceptual experiments; or adequate criteria may not have been employed in the selection of odors used in the electrophysiological experi­ ments to test the hypothesis. In view of the complexity of the problem of dealing with the coding of odor quality, certain basic principles related to the coding of odor intensity and duration are emerging. In a typical neurophysiological experiment, about 10 to 36 different odors are presented to each olfactory receptor neuron to test its responsivity. The saturated vapor is typically diluted 10-fold to 100-fold prior to presentation. Because of the differences in vapor pressures, the actual concentration in molar units ( M ) can range from 10" to 10" M or lower for compounds with very low vapor pressures like the musks. Hence, it is important to note that all odors are not presented to each receptor neuron at the same concentration. Thus, a bias in favor of the odors with the higher vapor pressures is built into the selection of responsive units. Regardless of this consideration, about 30 to 50 % of the cells are responsive to at least one odor (for review see Gesteland, 1976; Holley and MacLeod, 1977). Many of these neurons may be responsive to more than one odor. In general, knowing the response of a receptor unit to one odor, it is difficult to predict its response to a second one. Molecular structure, functional groups, physicochemical properties, and psychological impressions evoked by the odor, in addition to the experimenter's ability to define what is a "response," are undoubtedly critical factors. In response to well-controlled and monitored stimulus pulses, the discharge patterns recorded from olfactory receptor neurons are well defined. Inspec­ tion of Fig. 5 shows an excitatory discharge (top) recorded from a receptor neuron in response to a monitored odor pulse of n-butanol, 1 s in duration. When quantitative techniques of odor delivery are employed (Kauer, 1974; Kauer and Moulton, 1974; Kauer and Shepherd, 1975 ; Getchell and Getchell, 1977a; Getchell and Shepherd, 1978a), the odor pulse has a nearly square form with a very sharp onset, a rapid rise to peak concentration, and an abrupt turn-off at the end of the pulse, as is shown by the monitor (bottom). 4

7

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1. Olfactory Neurophysiology

Spike

potentials

Electroolfactogram

ÔÔ

Monitor

C0

2

L

Analyzer

Olfactometer

Fig.

5.

Schematic drawing o f the experimental set-up, showing the relations o f the o d o r

delivery nozzle from the stimulus delivery system and the recording electrodes to the surface o f the ventral olfactory epithelium o f the salamander. recorded spike potentials (top)

T h e traces show the

simultaneoulsy

from an olfactory receptor neuron in response to an o d o r and the

monitor ( b o t t o m ) of the o d o r ' s profile at the stimulus delivery nozzle tip located in close proximity to the recording site. ( F r o m Getchell and Shepherd, 1978a.)

The excitatory discharge of impulses is clearly related in the temporal domain to the shape of the odor pulse. The time delay between the onset of the monitor and the first spike is the latency of the response and has a duration of approximately 500 ms in this case. The impulse discharge consists of nine action potentials that have a characteristic pattern. As the discharge proceeds, the time between the impulses (the interspike interval) decreases in duration until about midway through the response. The interspike intervals then become progressively longer following the termination of the pulse. This precisely timed impulse discharge is called the phasic response property of the receptor cell. It closely resembles the phasic properties of other sensory receptors, e.g., cutaneous thermal receptors and certain neurons in the central nervous system. As the concentration of odor is changed, there are very

14

Thomas V . Getchell and Marilyn L . Getchell

precise and predictable changes in the characteristics of the impulse discharge. The threshold of a responsive receptor neuron is difficult to define quanti­ tatively for essentially three reasons. First, it has proven extremely difficult to determine empirically the actual concentration of odors interacting with the chemoceptive membrane. The use of quantitative techniques of olfacto­ metry in certain laboratories (e.g., those of J. C. Kauer, G. M. Shepherd, T. V. Getchell, and A. Holley) have yielded the concentration in molar units or molecules/ml of the odor at the stimulus delivery nozzle tip. How the proper­ ties of the overlying mucus layer affect the concentration, distribution, and retention of molecules is not accurately known but is currently an area of active investigation (e.g., Getchell and Getchell, 1977a,b; Hornung and Mozell, 1977). Second, since the spontaneous activity recorded from ol­ factory receptor cells is quite low, typically less than 25 spikes/min and irregular, only statistical procedures can reveal changes in activity. Fortunately, this approach is being pursued (Getchell, 1974a ; Van Drongelen, 1978). Third, since intracellular recordings are difficult to obtain routinely, the time course of the transmembrane voltage events prior tö the initiation of spike electrogenesis is not accurately known. In an initial study, Getchell (1977a) estimated the duration of these events to range from approximately 60 to 570 ms. Hence, because of these considerations, threshold is opera­ tionally determined by the experimenter's subjective criteria as being a just noticeable difference in the impulse discharge of an olfactory receptor neuron when short-duration odor pulses are delivered to the olfactory epithelium. Figure 6 shows recordings (a) from an olfactory receptor neuron responsive to the odor anisole and the instantaneous frequency plots of the excitatory discharge (b). The unit had an irregular pattern of spontaneous activity [Fig. 6(a), 1 and 2] with a mean interspike interval of about 1 s. There was no obvious change in activity at a concentration of 1.8 χ 10" M anisole (3), but a just perceptible change (4) was evoked by 3.6 χ 10" M anisole. Physiological threshold was estimated to lie between 1.8 and 3.6 χ 10" M anisole. As the concentration of anisole was increased, more vigorous excitatory responses were elicited (5 and 6). Each response was followed by a period of impulse inactivity lasting approximately 2.5 s. At each concen­ tration, the excitatory discharge and the period of impulse inactivity was related to the odor pulse in a very precise temporal manner. Figure 6(b) shows that with increasing odor concentration: one, the latency ( O ) of the impulse discharge decreases; two, there is a systematic relationship between peak firing frequency and odor concentration ; three, there is an apparently faster rate of initial impulse discharge; and four, the phasic discharge of impulses consists of an orderly array of interspike intervals. 7

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1. Olfactory Neurophysiology

»

15

1

Is F i g . 6.

T h e traces (left, a ) show samples o f a receptor neuron's spontaneous activity ( 1 , 2 )

and its response ( 3 - 6 ) to increasing concentrations of the o d o r anisole; instantaneous frequency plots (right, b ) o f the excitatory responses (left, 4 - 6 ) show a systematic relationship between the parameters o f the response and o d o r concentration. ( F r o m Getchell and Shepherd, 1978a.)

Certain receptor neurons are responsive to two or more odors, as is shown in Fig. 7. The responses shown as instantaneous frequency plots (left) were evoked by just-over-threshold concentrations of 3.7 μΜ nitrobenzene ( # ) and 4.5 μΜ benzaldehyde ( O ) . Each discharge consists of an orderly array of impulses which follow the basic pattern established for excitatory responses (Getchell, 1974a; Getchell and Shepherd, 1978a). Although the durations of the excitatory discharges were approximately equal (about 1.6 s), it is

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Thomas V . Getchell and Marilyn L . Getchell

(1//)

T

Fig. 7.

i

m

e

is)

Interval

d u r a t i o n (ms)

T h e instantaneous frequency plots (left) show the basic parameters o f the excitatory

responses e v o k e d in the same olfactory receptor neuron by just-over-threshold

concentrations

o f nitrobenzene ( # ) and benzaldehyde ( O ) . T h e distribution o f interspike intervals around the mean interval (x) for each excitatory response is shown on the right.

apparent that nitrobenzene evoked a more vigorous excitatory discharge than benzaldehyde. Both the peak firing frequency ( # , 25 impulses/s; O , 12 impulses/s) and the mean rate of discharge ( # , 13 spikes/s; O, 7 spikes/s) were greater in the response evoked by nitrobenzene. The responses also differed in the duration of their mean interspike intervals ( # , χ = 60 ms; Ο, χ = 130 ms) and the distribution of intervals about the mean, as is shown in the accompanying histograms (Fig. 7, right). These observations leave little doubt that both compounds activated excitatory processes in the same receptor cell. Thus, by general criteria the receptor neuron did not discrimi­ nate between nitrobenzene and benzaldehyde, which implies that the same molecular recognition mechanism was employed, since both compounds evoke similar almond odor sensations. Yet these interpretations are equivocal. The responses have different statistical properties, which suggests that the receptor cell is distinguishing between the two stimuli at similar concentra­ tions. Clearly, carefully constructed single-unit recording experiments and specific quantitative analyses of impulse discharges are required in order to be reasonably certain whether or not a receptor neuron discriminates between stimuli as distinct from questions related to molecular discrimination.

17

1. Olfactory Neurophysiology

Certain first principles have been derived from electrophysiological recordings from olfactory receptor neurons in response to changing odor concentration. The threshold, although difficult to estimate, for different receptor cells to the same odor may range from 10" to less than 10" M . The threshold of the same receptor neuron to different odors may be within the same order of magnitude or can vary by as much as five log units. This may reflect differing numbers of molecular receptor sites on the chemoceptive membrane or receptor sites of differing sensitivity. There is a systematic relationship between the parameters of the excitatory discharge and the concentration of the odor. When quantitative techniques of olfactometry are employed, there is apparently no distinctive pattern of impulses during an excitatory discharge to identify the odor type, molecular species, or perceived odor quality of the stimulus. The term "apparent" is employed 3

10

9

α 3s

Αν\/·ν*

_ ιο

^ b

10

c

5s

7s

5 0 ι.ο

d 9s

10 Time (s) Fig. 8 .

T h e trace (inset, right) shows an excitatory response recorded from an olfactory

receptor neuron in response to a 3-s pulse o f estragole at 3.9 χ 1 0 "

8

M. T h i s and subsequent

responses e v o k e d by increasing the duration o f stimulation from 3 to 9 s are shown as instan­ taneous frequency plots (left, a - d ) . N o t e in the responses recorded during the longer duration o d o r pulses ( b - d ) that each response consists o f distinct initial phasic and later tonic components. ( F r o m Getchell and Shepherd, 1978b).

18

Thomas V . Getchell and Marilyn L . Getchell

because of the considerations described above and also simply because not every possible odor can be applied systematically to each receptor neuron during electrophysiological experiments. In addition to changes in odor concentration, olfactory receptor cells transmit information about the duration of exposure. Quite simply, most receptor cells do so by transmitting a steady discharge of impulses to the brain throughout exposure to the odor (Getchell and Shepherd, 1978b). For example, the responses shown in Fig. 8 were evoked by just-over-threshold concentrations of 3.9 χ 10" M estragole. As the duration of stimulation was increased from 3 s (a) to 9 s (d), each response consisted of an initial phasic component that attained a peak discharge of about 10 impulses/s and a later tonic component at a frequency of about 6 impulses/s. The tonic rate of discharge was remarkably stable throughout the longer duration odor pulses. Hence, olfactory receptor cells are classified as slowly adapting receptors along with certain mechano- and thermoreceptors. From introspective experience, one realizes that odor perception decreases rapidly upon prolonged exposure. Hence, in contrast to visual perception, olfactory perception would be rapidly adapting. Although earlier electro­ physiological recordings of the electroolfactogram (Ottoson, 1956) suggested that olfactory receptor neurons are slowly adapting, there is the widely held belief that the major component of perceptual odor adaptation is due to the rapidly adapting properties of olfactory receptor neurons. The results of Getchell and Shepherd (1978b) as described above clearly indicate that most olfactory receptor neurons are slowly adapting in response to prolonged odor stimulation. Presumably, more central neural mechanisms modulate the incoming signals carried by the olfactory receptor neurons during the tonic discharge so as to account for the reduced perceptual impressions of the odor. The neural basis of this phenomenon may be attributed, at least in part, to inhibitory synaptic mechanisms in the olfactory bulb as described by Getchell and Shepherd (1975a,b), Kauer and Shepherd (1977), and Getchell and Shepherd (1978b). In summary, neurophysiological investigations of the olfactory epithelium have revealed a complexity of basic membrane mechanisms and principles of neural coding that were not anticipated in view of its relatively simple neuronal organization. It appears that different receptor neurons have different molecular receptors located on the cilia and/or knob, apparently detecting a narrowly defined group of odors. The olfactory receptor cell's view of the odor world apparently does not conform with psychologically constructed categories of primary odors. Therefore, the neural mechanisms for the coding of odor quality remain a fascinating but enigmatic topic of discussion and experimentation. The sequence of neural events that leads to the transmission of sensory information to the brain has been carefully 8

1. Olfactory Neurophysiology

19

outlined and documented, but numerous fascinating specific details remain unexplored (Getchell, 1977a). Certain principles of sensory coding by olfactory receptor neurons related to odor intensity and duration have been elucidated. Depending on the duration of the odor pulse, the responses recorded from the receptor neuron have distinctive phasic and tonic com­ ponents. Olfactory receptor neurons are slowly adapting receptors, as is shown by their response properties to relatively long duration odor pulses. The relevance and implications of these first principles for considerations related to the coding of odor quality and psychologically defined odor sensations are becoming apparent.

III. MOLECULAR BIOLOGY OF ODOR DETECTION

The ability of olfactory receptor neurons to discriminate among odors, man's ability to distinguish perceptually among numerous odors, and studies of specific olfactory deficits (e.g., Amoore, 1977a; Wysocki et al, 1977) strongly support the hypothesis that molecular receptor molecules are integral components of the chemoceptive membrane. The receptor molecules are envisioned to have properties similar to those described for acetylcholine receptors on the subsynaptic membrane of the neuromuscular junction, certain hormone receptors in the endocrine system, and active sites of enzyme molecules. Numerous attempts have been made over the last several years, employing biochemical techniques, to isolate receptor molecules from the receptor cell membrane and to investigate their binding properties. Partially purified "protein" extracts have been prepared from olfactory epithelial tissue. Several assays have been employed to examine the binding properties of odors with the macromolecular fraction. They include : changes in absorbance in the ultraviolet spectra (Ash, 1968; Gusel'nikov et al, 1974); changes in enzyme activities (Ash, 1969; Koch and Desaiah, 1974); changes in conductance in reconstituted lipid membranes (Cherry et al, 1970; Fesenko et al, 1977); binding of radiolabeled odorants (Gennings et al, 1977); and affinity chromatography (Price, 1978). These studies suggest that a protein is the recognition molecule that initially interacts with odors. In general, control tissue taken from nearby respiratory epithelium and mucus does not exhibit a similar activity. Further efforts to isolate specific cell types in the olfactory epithelium prior to subcellular fractionation and protein isolation are required in order to take full advantage of these approaches. In addition, the results of Getchell and Gesteland (1972), who employed group specific protein reagents coupled with an electrophysiological assay, further indicate that sulfhydryl groups are involved at the recognition site on the receptor molecule. The approaches of biochemistry and molecular biology

20

Thomas V . Getchell and Marilyn L . Getchell

(e.g., Bannister et al, 1975; Margolis, 1977; Price, 1978) hold promise for characterizing the receptor molecule, its binding properties, and the molec­ ular organization of the peripheral olfactory system.

IV. SUMMARY: SCHEMA OUTLINING THE SEQUENCE OF EVENTS LEADING TO THE TRANSMISSION OF OLFACTORY INFORMATION TO THE BRAIN

Sensory information about the odor world is transmitted to the brain by olfactory receptor neurons through an orderly sequence of molecular, membraneous, and neural events. The processes occurring nearly simul­ taneously in numerous receptor cells and subsequently in neurons in the brain are the substrates on which odor perception is presumably constructed. The processes are initiated (Fig. 9) by the impingement of odor molecules with concentration [ S ] on the surface of the olfactory mucus. The concentra­ tion of stimulus molecules in the headspace above the mucus may be cal­ culated from the ideal gas laws and empirically determined using gas chromatographic techniques (O'Connell and Mozell, 1969; Poynder, 1973; Kauer, 1974; Bostock, 1974; Revial et al, 1978). The concentration of the stimulus in mucus [ S ] at the air-mucus interface is a function of the par­ tition coefficient B: f

m l

Β = [S] /[S] f

m l

[ S ] and, hence, the factor Β have not been empirically determined for any odor in olfactory mucus. Olfactory mucus consists of two distinct physical systems, a superficial watery layer overlying a thicker mucoid layer. The former layer may be derived from secretions released by the subepithelial Bowman glands (Reese, 1965; and Brightman, 1970; Seifert, 1971; Breipohl, 1972). Possible functions and release mechanisms have been discussed by Getchell and Getchell (1977a,b). The latter layer may be derived from the sustentacular cells (Graziadei, 1971; Okano and Takagi, 1974; Getchell, 1977a,b). Exocytotic release mechanisms, identification and plausible func­ tions have also been proposed by Getchell and Getchell (1977a,b). The olfactory cilia are reported to lie at the interface of the two systems (Reese, 1965). It is assumed that certain stimulus molecules must traverse the super­ ficial mucus layer to interact with the distal ciliary membrane, while others must traverse both mucus layers prior to molecular interaction. Hence, it may be necessary to use a second partition coefficient, B to describe the concentration of molecules at the watery-mucoid-mucus interface: m l

l9

*i = ( [ S ] * - * ) / [ S ] where χ represents that part of [S] * that interacts with receptor sites located on the olfactory cilia at the watery-mucoid-mucus interface. 3

3

m 2

ο σ

Ο ο eu ν , ra

ο φ c

Jo

< <υ ο ο «-< se tin

1 * ' υ ϊ

φ ε φ

ο ο Ε

(Ο

φ

α: -

+

Ε

/Τ ο

ο

2

ο

Ο

ω

Ή

.Ο

"δ φ

ε

>

c φ

Φ

ΤΗ·*

to -

α 2.

Ε ι—I CO

τ

CO

β

—,

Ο£

S sd

M

Ο

'S g cä cd ε £ .S ε

22

Thomas V . Getchell and Marilyn L . Getchell

Perireceptor processes functioning in the mucus may also determine the penultimate concentration of the stimulus [ S ] * and [ S ] , and its spatialtemporal profile in the region of the chemoceptive membrane. These pro­ cesses may be isolated for heuristic purposes and described by the rate constants / c , / c , and k for those occurring in the mucoid mucus and k%, k%, k\ for similar processes occurring in the watery mucus. The main process controlling access of molecules to the membrane is diffusion through each layer of mucus. Assuming that each layer is a stationary isotropic system, diffusion would be ideally described by Fick's law and described by the rate constants /c and k%. Diffusion times have not been determined, but estimates ranging from 100 ms to 1-2 s have appeared in the literature (Ottoson, 1970). Mucus is not stationary. It is being constantly replenished and swept over the epithelial surface toward the internal naris at rates estimated to range from 10 to 60 mm/min (Moulton and Beidler, 1967). Hence, the rate constants fc and /cf represent processes by which molecules [S] are swept away from a spatially restricted region by mucociliary transport. Numerous enzymes have been identified in the cellular elements (reviewed by Getchell and Getchell, 1977b). These may well function either intracellular^ or extracellularly to enzymatically degrade certain odor molecules prior to or subsequent to their interaction with the chemoceptive membrane. These and other chemical processes, identified by the rate constants fcf and / c , also contribute to shaping the spatial-temporal concentration profile of odor molecules in mucus leading to [ P ] . Thus, partition coefficients and several perireceptor processes occurring in mucus may play primary roles in controlling the access of odors to and presumably their egress from the chemoceptive membrane. A receptor site may be operationally defined as a portion of a molecule R capable of binding one or more stimulus molecules S. Most importantly, the resulting S-R complex is coupled to specific ion conductance mechanisms across the receptor cell membrane, which initiates the orderly sequence of electrical events in the receptor cell previously described. The interaction of S with R will thus result in a transmembrane voltage change, the generator potential, which, if of sufficient magnitude, will activate spike electrogenic mechanisms in the somatic membrane (Getchell, 1973, 1977a,b). The molecular interaction of S molecules with membrane molecules is a prob­ abilistic function described by the law of mass action, largely depending on [ S ] and [ S ] . It will involve both specific S-R interactions and non­ specific molecular interactions with structural membrane molecules. Hence, the total number of stimulus molecules specifically interacting with R molecules may be defined as [ S ] . The rate constants k and k describe the formation of the S-R complex and the reverse reaction. There is little evidence 3

4

3

m 3

2

4

3

2

m 3

m 3

m 4

a

ß

23

1. Olfactory Neurophysiology

as to the nature of R, but as described above, it is probable that R is a site on a protein molecule that specifically interacts with S. Thus, the multifaceted approach employing neuroanatomical, electro­ physiological, biochemical, and molecular biological techniques is beginning to unravel the sequence of molecular, membraneous, and neural events underlying man's perceptual impressions of the marvelously rich odor world.

ACKNOWLEDGMENT T h e authors were supported by grants from the N a t i o n a l Science Foundation ( B N S - 1 2 6 0 1 ) and the N a t i o n a l Institutes o f Health ( N I N C D S - N S - 1 6 3 4 0 ) during the writing o f this contri­ bution. W e thank R o b e r t Erickson, Frank M a r g o l i s , Steven Price, and an unknown reviewer for their comments on the manuscript.

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