Sensory Neurons

Olfactory sensory neurons located in the nasal epithelium detect specific odorants and respond by sending action potentials to the brain (Fig. 27-1). These neurons have three specialized zones. An apical dendrite extends to the surface of the epithelium and sprouts approximately 12 sensory cilia specialized for responding to particular extracellular odorants. The response depends on high concentrations of four proteins in the ciliary membrane: a single type of odorant receptor, the trimeric G-protein G_olf, type III adenylyl cyclase, and cyclic nucleotide–gated ion channels. The cell body contains the nucleus, protein-synthesizing machinery, and plasma membrane pumps and channels that set the resting electrical potential of the plasma membrane. An axon projects from the base of each neuron to secondary neurons in the olfactory bulb at the front of the brain.

Figure 27-1 olfaction. A, Light micrograph of a section of sensory epithelium from the nasal passage of a mouse after staining (purple) for one olfactory receptor mRNA. Note that only a few cells express this gene. B, The sensory epithelium, highlighting one olfactory sensory neuron and the signal-transducing proteins concentrated in three parts of the cell. (BM, basement membrane.) C, Signal transduction mechanism. An odorant molecule binds a specific seven-helix plasma membrane receptor and changes its conformation. The activated receptor catalyzes the exchange of GDP for GTP on multiple trimeric G-proteins, causing dissociation of G_olfα from Gβγ. G_olfα-GTP activates adenylyl cyclase to produce multiple cAMPs. cAMP binds to and opens cyclic nucleotide–gated ion channels in the plasma membrane, depolarizing the plasma membrane. Ca²⁺ admitted by the cAMP-gated channel opens chloride channels (ClC), which augment membrane depolarization. Membrane depolarization triggers an action potential at the base of the axon that travels along the axon to secondary neurons in the brain. Multiple negative feedback loops (red) terminate stimulation. cAMP activates PKA, and Gβγ activates odorant receptor kinase (ORK [green]), both of which phosphorylate and inhibit the receptor. Ca²⁺ binds calmodulin, which inhibits the cAMP-gated channel and activates phosphodiesterase (PDE) to break down cAMP. D, Light micrograph of the olfactory bulb of a mouse expressing a marker enzyme (beta galactosidase) driven by the promoter for one odorant receptor. A histochemical reaction marks the axons of these cells blue. Axons from many olfactory sensory neurons expressing the same receptor converge on the same glomerulus in the olfactory bulb.

(A, From Ressler KJ, Sullivan SL, Buck LB: A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73:597–609, 1993. D, Courtesy of Charles Greer, Yale University, New Haven, Connecticut. Reference: Zou D-J, Feinstein P, Rivers AL, et al: Postnatal refinement of peripheral olfactory projections. Science 304:1976–1979, 2004.)

Olfactory sensory neurons are unique among adult neurons in their ability to replace themselves from precursor cells in the epithelium within 30 days after they are destroyed. If protected from viruses and environmental toxins, they turn over much more slowly, so the ability of self-renewal appears to be an adaptation to the hazards associated with exposure to the environment. Loss of olfactory function with age results, in part, from a decreased ability to maintain this neuronal replacement process. The presence of neurons in an epithelium might seem odd, but recall that the entire central nervous system derives from the embryonic ectoderm.

Overview of the Pathway

Extracellular odorants bind to receptors on the cilia of olfactory neurons, depolarize the plasma membrane, and initiate action potentials at the base of the axonal process. The process of reception and transduction of stimuli takes place in five steps:

As you can appreciate from everyday experience, olfactory signal transduction is very sensitive but adapts quickly to the continued presence of an odorant. An understanding of each step in the sensory transduction and adaptation pathways helps to explain how animals discriminate among so many different odors.

Odorant Receptors

The olfactory system uses a large family of seven-helix receptors to detect a wide range of ligands present at low concentrations in mucus. These receptors were identified by cloning their complementary DNAs (see Fig. 6-8 for cDNAs) from the olfactory epithelium. Genome sequencing established that mice have about 1000 functional odorant receptor genes (about 4% of total genes!), humans have about 350 functional genes, and fish have 100. Odorants are presumed to bind among the transmembrane helices, the most variable part of these proteins. Each sensory neuron typically expresses a single type of odorant receptor (Fig. 27-1A), using negative feedback from the receptor itself to suppress the expression of other types of odorant receptors. The 1000 cells that express each receptor are scattered in zones throughout the olfactory epithelium.

G-Protein Relay

Odorant binding changes the conformation of the receptor, allowing it to catalyze the exchange of nucleotide on G_olf on the cytoplasmic face of the plasma membrane. In less than 100μs, an activated receptor can produce 10 to 100 activated GTP-G_olfα molecules, which then dissociate from their Gβγ subunits.

Production of cAMP

In the third step, GTP-G_olfα binds to and activates a specialized olfactory isozyme of adenylyl cyclase. While active, each enzyme generates nearly 100 cAMP molecules. By 75μs after stimulation, the concentration of cAMP inside the cilium peaks at >10μM, returning to baseline within 500μs. This dramatic fluctuation in cAMP concentration is made possible by the high surface-to-volume ratio of cilia, which ensures that membrane-associated signaling components interact rapidly and confines the cAMP within a small volume. The cAMP transient is short-lived, owing to hydrolysis by a phosphodiesterase with a high turnover rate but a relatively low affinity for cAMP.

Cyclic Nucleotide–Gated Channels Depolarize the Plasma Membrane and Trigger an Action Potential

The fast cAMP transient depolarizes the plasma membrane by activating cyclic nucleotide–gated cation channels. The concentration of these channels in ciliary membranes (>2000/μm²) is much higher than that in the cell body (6/μm²). Binding of at least two cAMP molecules increases the probability that the channel is open from near 0 to about 0.65. Because the probability is not 1.0, individual activated channels flicker open and closed on a millisecond time scale. The ensemble of many activated channels admits enough Na⁺ and Ca²⁺ to depolarize the membrane. Ca²⁺-activated chloride channels carry additional current. The lag of 200 to 500μs between binding of the odorant and peak membrane depolarization is attributable to the relatively slow binding of cAMP to the channel.

The role of cAMP in olfactory signaling was established by experiments on isolated olfactory neurons, in which the effects of odorants, membrane-permeant cyclic nucleotide analogs, and phosphodiesterase inhibitors were explored. Null mutations in mice confirmed the importance of cAMP-gated channels. Note that the role of cAMP in olfaction is distinctly different from its role in most other tissues, where the main target of cAMP is protein kinase A (PKA [see Fig. 25-3]).

Depolarization of the ciliary membrane initiates an action potential (see Fig. 11-6) by activating voltage-gated sodium channels (see Fig. 10-2) in the cell body. The action potential propagates along the axon to a chemical synapse with the second neuron in the olfactory bulb of the brain. The two stages of amplification downstream of the receptor allow a few active receptors to produce an action potential.

Adaptation

Desensitization—the waning of perceived odorant intensity despite its continued presence—results from a combination of central and peripheral processes. Peripheral processes that contribute to adaptation include modulation of each step in the signaling pathway following odorant binding (Fig. 27-1C). At the molecular level, this adaptation is reflected in the transient nature of G-protein activation, the self-limited increase in cAMP, and the limited duration of the membrane depolarization, all of which occur with constant exposure to odorant. Importantly, the sequential nature of many of these feedback circuits implies that they have intrinsic delays and therefore serve not only to alter the magnitude of the response but also to shape its time course.

G-protein-coupled receptors are desensitized by protein kinases that phosphorylate the receptor and by proteins called arrestins that bind phosphorylated receptors (see Fig. 24-3). These modifications inhibit the interaction of activated receptors with G-proteins and provide negative feedback at the first stage of signal amplification. Negative feedback is coupled to receptor stimulation, because the olfactory receptor kinase is brought to the plasma membrane by binding the Gβγ subunits released by receptor-induced G-protein dissociation.

Ca²⁺ entering the cell through cyclic nucleotide–gated channels binds calmodulin, and the complex provides negative feedback at two levels. Calcium-calmodulin activates the cAMP phosphodiesterase, which rapidly converts cAMP to 5′-AMP. Calcium-calmodulin also binds to the cyclic nucleotide–activated channel, reducing its affinity for cAMP by 10-fold. This change reduces the probability of the channel’s opening at less than saturating cyclic nucleotide concentrations and might accelerate the otherwise slow dissociation of cAMP from the channel. These two effects of Ca²⁺ alter the responsiveness of the cell to initial odorant exposure, shape the time course of the response, extend the dynamic range over which the cell can respond, and make a cell transiently refractory to additional stimulation.

Processing in the Central Nervous System

Mammals discriminate many more odorants than the number of available receptors by combining information from multiple types of receptors in their central nervous systems. While the sensory neurons that express any single odor receptor are broadly distributed across the olfactory epithelium, the axons from this family of like sensory neurons converge on only two to three targets in the olfactory bulb. The target, a glomerulus, is a dense area with synapses between axons of olfactory sensory neurons and dendrites of the second neurons in the pathway. Because each glomerulus receives input only from axons that express the same odor receptor, the molecular specificity established in the olfactory epithelium is preserved. Given approximately 1000 odor receptors in the mouse, each mouse olfactory bulb has approximately 2000 glomeruli (Fig. 27-1D). Of special interest, the odor receptor itself is an important determinant of axon targeting to the glomeruli. Substitution of odor receptors results in the axons selecting new glomerular targets. About 50 secondary neurons receive synaptic input within a glomerulus. Most of these neurons send their axons to higher levels, where they terminate in a combinatorial manner on cortical neurons.

The discrimination of a particular odorant is achieved in two stages: At the first stage, each odorant activates several different receptors, and each receptor can bind a group of related odorants. Therefore, each odorant activates a particular pattern of olfactory sensory neurons and their coupled glomeruli. At the next level, neurons in the cerebral cortex receive information from a combination of glomeruli, leading to eventual discrimination of many different smells at higher levels of the brain. See Box 27-1 for information on our second olfactory system.

Overview of Visual Signal Processing

Photons are energetic but unconventional agonists. They are tiny, move very fast, and penetrate most biochemical materials. These properties create a formidable challenge for detecting photons and transducing their properties (intensity and wavelength) into a signal that can be transmitted to the brain. Nevertheless, vertebrate photoreceptor cells capture single photons and convert this energy into a highly amplified electrical response (Fig. 27-2). Phototransduction is the best-understood eukaryotic sensory process because the system is amenable to sophisticated biophysical, biochemical, and physiological analysis. Single-cell organisms use similar mechanisms to respond to light (see Fig. 38-19).

Figure 27-2 vertebrate visual transduction. A, Drawing of a rod cell. Disks in the outer segment are rich in rhodopsin. B–D, Drawings of small portions of an outer segment (upper panels) and the synaptic terminal of a rod cell (lower panels) in three physiological states. Active components are highlighted by bright colors. B, Resting cell in the dark. Constitutive production of cGMP keeps a subset of the plasma membrane cGMP-gated channels open most of the time, allowing an influx of Na⁺ and Ca²⁺. At the resting membrane potential, the synaptic terminal constitutively secretes the neurotransmitter glutamate. Ca²⁺ leaves the outer segment via a sodium/calcium exchange carrier in the outer segment, whereas Na⁺ leaves the cell via a sodium pump in the plasma membrane of the inner segment. C, Absorption of a photon activates one rhodopsin, allowing it to catalyze the exchange of GTP for GDP bound on many molecules of transducin (G_T). This dissociates G_Tα from Gβγ. Each G_Tα-GTP binds and activates one molecule of phosphodiesterase (attached to the disk membrane by N-terminal isoprenyl groups), which rapidly converts cGMP to GMP. As the concentration of free cGMP declines, the cGMP-gated channels close, leading to hyperpolarization of the plasma membrane and inhibition of glutamate secretion at the synaptic body. D, Recovery is initiated when rhodopsin kinase phosphorylates activated rhodopsin. Binding of arrestin to phosphorylated rhodopsin prevents further activation of G_T. Phosphodiesterase and an RGS protein cooperate to stimulate hydrolysis of GTP bound to G_T, returning G_T to the inactive G_Tα-GDP state. Synthesis of cGMP by guanylyl cyclase returns the cytoplasmic concentration of cGMP to resting levels and opens the cGMP-gated channels. Constitutive secretion of glutamate resumes.

Vertebrate photoreceptor cells are neurons located in a two-dimensional array in the retina, an epithelium inside the eye. The cornea and lens of the eye form an inverted real image of the outside world on the retina, so the intensity of the light across the field of view is encoded by the array of geographically separate photoreceptor cells. Photoreceptor cells lie at the base of a complex neural processing system. Having detected the rate of photon stimulation at a particular place in the visual field, photoreceptor neurons communicate this information to higher levels of the visual system. Initial processing of the information takes place in the retina, where secondary and tertiary neurons take input from multiple photoreceptors to derive local information regarding image contrast, as well as color and intensity. Neuroscience texts present more detailed information on higher levels of visual processing in the retina and brain.

The response of photoreceptor cells depends on the intensity of the light, that is, the flux of photons. Vertebrate retinas detect light with intensities that range over 10 orders of magnitude. Rod photoreceptors (Fig. 27-2A) detect low levels of light from about 0.01 photon per μm²per second (dim stars) to 10 photons per μm²per second but do not discriminate light of different colors. Cone photoreceptors (cones) respond to more intense light, up to about 10⁹photons per μm²per second (full sunlight). Three classes of cones with chromophores that are sensitive to different wavelengths of light allow humans to encode wavelength and color vision to operate (Box 27-2).

Rods and cones have three specialized regions with different molecular components and functions. The nucleus and the organelles in the inner segment maintain the cell’s structure and metabolism. A vestigial cilium connects the inner segment to the outer segment, which consists of a stack of internal membrane disks containing the photoreceptor protein, rhodopsin in the case of rods, surrounded by the plasma membrane. Disks form by invagination and pinching off of flattened sacks of plasma membrane. Rhodopsin synthesized in the cell body is transported to the plasma membrane along the secretory pathway and segregated into disk membranes. The lumen of the disks corresponds topologically to the lumen of the endoplasmic reticulum or the extracellular space.

Absorption of a photon activates rhodopsin and initiates a signaling cascade (Fig. 27-2) involving a trimeric G-protein and a cGMP phosphodiesterase, both attached to the cytoplasmic face of the disk membrane by covalent lipid groups. Active phosphodiesterase lowers the cytoplasmic concentration of cGMP and closes cGMP-gated channels in the plasma membrane. Closing these channels reduces the release of glutamate at the synapse with the next neuron in the visual circuit. Signals flow through the system as follows:

Feedback loops operate at every level in this signal transduction pathway, turning off the response to a flash of light. The following sections explain how these reactions achieve their spectacular sensitivity in rods.

Rhodopsin

Rhodopsin, the photoreceptor protein of rods, is a seven-helix, G-protein-coupled receptor with a light-absorbing chromophore, 11-cis retinal, covalently attached to lysine 296 through a protonated Schiff base (see Fig. 24-2B). Although 11-cis retinal is bound to a site in the bundle of transmembrane helices similar to sites where ligands bind other seven-helix receptors, this form of rhodopsin is inactive with respect to catalyzing nucleotide exchange on its trimeric G-protein. Thus, rhodopsin is a seven-helix receptor with a covalently attached, but inactive, ligand.

The ability of rods to detect single photons depends on two favorable properties. First, the noise level is very low, owing to the stability of the 11-cis retinal. In vertebrate rods, fewer than one molecule in 4×10¹⁰isomerizes spontaneously every second, so the background level of activated rhodopsin is very low, even with more than 10⁸molecules of rhodopsin per cell. Thus, one does not see spots of light in the dark. Second, rods absorb photons very efficiently by virtue of the high concentration of rhodopsin in disks (∼25,000 rhodopsins/μm²) and stacking thousands of disks on top of each other in the direction of incoming photons. Rhodopsin constitutes 90% of the disk membrane protein and 45% of the disk membrane mass. About half of the photons that traverse the outer segment are absorbed, and about two thirds of absorbed photons produce an electrical change in the plasma membrane.

Absorption of light initiates the signal transduction pathway. Picoseconds after the 11-cis retinal chromophore absorbs a photon, the energy isomerizes it to all-trans retinal. This change initiates a cascade of intramolecular reactions that activates rhodopsin by changing its conformation. Metarhodopsin II, the stable active conformation, has rearranged cytoplasmic loops that catalyze nucleotide exchange on transducin, its trimeric G-protein partner (see Fig. 25-9). The signal initiated by absorption of light is amplified by two successive enzymatic reactions and by closing ion channels. Following activation, rhodopsin is inactivated by hydrolysis of the Schiff base linking all-trans retinal to the protein and dissociation of the chromophore. Rhodopsin is regenerated by binding a fresh molecule of 11-cis retinal, derived from vitamin A.

The Positive Arm of the Signal Cascade

Metarhodopsin II catalyzes the exchange of GDP for GTP on the a subunit of transducin, which then dissociates its βγ subunits. Each metarhodopsin II produces hundreds of activated transducins in a fraction of a second, nearly as fast as the molecules collide while they diffuse in the plane of the very crowded disk bilayer. Nucleotide exchange on transducin is rate-limiting in the whole transduction cascade, even with a 10⁷acceleration by metarhodopsin II.

Transducin α-GTP activates phosphodiesterase by binding its two inhibitory γ subunits. This binding reaction does not amplify the signal but frees the catalytic α and β subunits of phosphodiesterase to break down cGMP to GMP at a high rate. The cytoplasmic concentration of cGMP depends largely on its rate of destruction by light-activated phosphodiesterase, as it is made continuously by guanylyl cyclase.

As the concentration of cGMP falls, cGMP-gated cation channels in the plasma membrane close. These channels (see Fig. 10-10) are very sensitive to the concentration of cGMP. Binding of four cGMPs opens a channel, whereas the loss of one cGMP closes a channel. Amplification in this pathway is spectacular. Within 1 second after absorption of a single photon, rhodopsin activates 1000 transducins and a similar number of phosphodiesterases, which break down 50,000cGMPs. This change in concentration closes hundreds of cGMP-gated channels, each of which blocks the entry of more than 10,000 cations. Box 27-3 provides more details about the electrical circuit in the rod cell.

Closure of cGMP-gated channels hyperpolarizes the membrane and inhibits glutamate release at the synapse. This light-induced decline in glutamate release has opposite effects on the two types of “bipolar neurons” connected to rods: It stimulates “on-type” bipolar neurons to fire action potentials and hyperpolarizes the “off-type” bipolar neurons. This combination of responses is the first step in the discrimination of contrast in our visual world.

Recovery and Adaptation

After a dim flash of light, the reduced cytoplasmic cGMP concentration and the plasma membrane hyperpolarization are short-lived, on the order of 2 seconds in rods, briefer in cones. Rods reset the signaling pathway by inhibiting metarhodopsin II, inactivating transducin α-GTP, and activating the synthesis of cGMP.

Rhodopsin kinase (now called GRK1 for G-protein-coupled receptor kinase 1), associated with the disk membrane by a C-terminal farnesyl group, phosphorylates several residues near the C-terminus of metarhodopsin II. Like other seven-helix receptor kinases, rhodopsin kinase is active only toward the active form of the receptor. Phosphorylation of rhodopsin reduces its ability to activate transducin. Binding of a second protein, arrestin, to phosphorylated rhodopsin prevents further production of transducin α-GTP.

GTP hydrolysis dissipates the light-activated burst in transducin α-GTP. The low GTPase activity of transducin is activated by association with phosphodiesterase and by an RGS protein (regulator of G-protein signaling; see Fig. 25-8), inactivating transducin in less than 1 second. Humans with mutations that disable the retinal RGS protein cannot adapt to rapid changes in light, so they are blinded for several seconds when they step out of a dark room into full sunlight. Dissociation of transducin α-GDP from phosphodiesterase inhibitory subunits terminates cGMP breakdown.

The reduction in cytoplasmic Ca²⁺ that accompanies closure of cGMP-gated cation channels stimulates the guanylyl cyclase that rapidly restores the cGMP concentration. This change opens the cation channels and returns the membrane potential to the resting level. See Box 27-4 for information on our second visual system.