Bi156 lecture 1/9/12. Control of sexual and social behaviors by the olfactory system

Transcription

1 Bi156 lecture 1/9/12 Control of sexual and social behaviors by the olfactory system

2 Anatomy of the mammalian olfactory system In many mammals (a rat shown here) the olfactory organs within the nose are split into the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). MOE neurons project to the main olfactory bulb (OB), while VNO neurons project to the accessory olfactory bulb (AOB). OB output neurons project to regions of cortex, while AOB output neurons project only to the amygdala.

3 Anatomy of main and accessory olfactory systems

4 MOE receptors and signal transduction There are >1000 genes in the rodent genome (>3% of total genes!) that encode olfactory receptors. These are 7-helix (G-protein coupled) receptors. Upon binding of the appropriate odorant compound, they activate the G s -like G protein G olf, whose α subunit activates adenylyl cyclase (AC). The camp thus generated opens a camp-gated cation channel, allowing Na + and Ca 2+ ions to flow into the neuron.

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6 Expression of MOE receptors The MOE contains several layers of ciliated olfactory receptor neurons (ORNs). ORN cilia contact volatile odorants adsorbed by the mucus overlaying the epithelium. Each ORN is thought to express only one receptor. Receptors can be divided into 4 groups, each of which is expressed only by neurons within one of the 4 expression zones within the MOE. Within one zone, neurons expressing a given receptor are randomly localized. ~1/250 of neurons within a zone express an individual receptor. Most ORNs are broadly tuned, and appear to be able to respond to a range of chemicals with related structures.

7 The VNO The VNO is thought to respond to pheromones (compounds that produce involuntary behavioral responses, most related to sex, pregnancy, aggression, and parent-offspring relations). Its structure (a pump with a liquid reservoir) allows it to respond to nonvolatile pheromones, but VNO neurons can also respond to volatile compounds. It is a cup-shaped organ near the front of the rodent nose; its neurons are divided into apical (near the lumen) and basal layers. The microvilli of the VNO neurons face the lumen. Neurons in the apical layer express the G protein α subunit G αi2, while those in the basal layer express G αo.

8 VNO anatomy and TRP2 (transduction) channel expression Expansion and contraction of the central vein is thought to allow the VNO to act like a pump, siphoning up pheromone-containing secretions into the lumen. The transduction channel and the receptors are located on the microvilli at the edge of the lumen (red).

9 VNO signal transduction VNO receptors are all thought to signal via activation of phospholipase C (PLC) by G protein βγ subunits. IP 3 and diacylglycerol generated by PLC then cause Ca 2+ entry through the microvillar VNO-specific TRP2 channel. This signaling pathway is much like that used in insect phototransduction.

10 VNO receptors There are 2 distinct families of VNO 7-helix receptors, all unrelated to MOE receptors. Each VNO neuron probably expresses only one receptor, as in the MOE. V2Rs (~60 functional genes in the mouse) are expressed in a random pattern by basal layer neurons (Go neurons). V2Rs have large N-terminal extracellular domains. V1Rs (~180 functional genes) do not have N-terminal extracellular domains. They are expressed by neurons within the apical layer (Gi neurons).

11 Humans do not have a VNO Examination of leftover noses from plastic surgery showed that some humans have a pit with neurons in the front of the nose. This had been interpreted as a VNO. However, all human V1R and V2R genes are pseudogenes, as is the human TRP2 channel gene. Thus, humans are unlikely to have a functional VNO, consistent with the fact that humans don t snuffle liquids into their noses.

12 Response characteristics of VNO neurons VNO neurons respond to urine. Some neurons selectively respond to urine from mice of the same sex, others to urine of the opposite sex. When tested with candidate pheromone compounds, VNO neurons can respond to very low concentrations. Unlike ORNs, their responses are narrowly tuned; no neurons were ever observed to respond to more than one compound. Mice produce ultrasonic calls ( whistling ) in response to contact with urine from the opposite sex; production of these calls requires the VNO.

13 Firing responses of female VNO neurons to male and female urine

14 Some pheromonal compounds The behavioral responses produced by these compounds are indicated. Some have been directly shown to stimulate firing of VNO neurons. Interestingly, the pig pheromones, although they are sex steroids and are not very volatile, are thought to be detected through the MOE and not the VNO (more on this later).

15 A map of ORN projections in the main olfactory bulb Although the MOE neurons expressing an individual receptor are randomly dispersed within an expression zone, all of these neurons project their axons to two glomeruli (balls of synapses surrounded by glia) within each olfactory bulb. Each bulb has ~2000 glomeruli in rodents, so there is an approximate correspondence between the number of glomeruli in each half-bulb and the number of receptor genes. This is a stereotyped map, and does not vary between individual animals.

16 Diagram of the projection map

17 Projections of VNO neurons to the AOB Neurons in the apical (G i ) layer of the VNO project to the rostral half of the AOB, while those in the basal (G o ) layer project to the caudal half. Neurons expressing closely related V1Rs can project to the same glomerulus. The AOB is divided into domains which contain glomeruli receiving input from related V1Rs. This projection pattern is not invariant, but is more similar between the two AOBs in one mouse than between AOBs in different mice. AOB mitral cells have dendrites that innervate multiple glomeruli in different domains that receive input from closely related V1Rs (selective heterotypic connectivity).

18 V1R AOB projection maps

19 MOB and AOB architectures Each MOB glomerulus receives input from neurons expressing only one receptor, and each mitral cell has a dendrite in only one glomerulus. Thus, the output from an MOB mitral cell represents the responses of a single receptor. In the AOB, neurons expressing a given receptor project to multiple glomeruli, and mitral cells have dendrites that arborize in multiple glomeruli. The output from an AOB mitral cell likely represents the responses to a group of related VRs.

20 Mitral cells in the MOB connect to multiple cortical areas.

21 AOB projections to the brain Mitral cells in the AOB have apical dendrites that arborize in multiple glomeruli. The AOB projects to the amygdala (directly), and the hypothalamus (via the amygdala). The projections from the rostral and caudal AOB halves are superimposed in the amygdala. This implies that integration of pheromone signals may take place primarily in the AOB.

22 VNO neural pathways Dye-filling experiments have shown that the VNO is connected to the medial preoptic area (MPOA) of the hypothalamus via the amgydala and BNST. No connections of the VNO to cortical areas were observed.

23 The main and vomeronasal olfactory systems have overlapping functions The old view suggested by projection maps is that the main olfactory system mediates cortical responses to volatile odorants, while the VN system mediates unconscious responses to water-soluble pheromone compounds found in urine and secretions of other individuals. GnRH (LHRH) neurons in the MPOA of the hypothalamus make a releasing hormone required for LH secretion and are necessary for mating and other behaviors associated with reproduction. They were thought to respond to VNO signals, since the VNO is connected to the MPOA. However, retrograde tracing studies using transgenic techniques show that GnRH neurons themselves are connected only to the MOE and not the VNO, suggesting that the inputs that drive unconscious responses in these hypothalamic neurons are stimulated by volatile compounds sensed by ORNs. Also, some pheromones (e.g. androstenone in pigs) are detected by the MOE, not the VNO. Behavioral analysis indicates that the MOE and VNO are both involved in control of mating and social interactions.

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25 Response characteristics of AOB neurons in intact, behaving animals Recordings were obtained from AOB mitral cells in awake, behaving mice. These mice were presented with anesthetized stimulus animals of various strains and sexes. AOB neurons fire during contact of the mouse s nose with the stimulus animal.

26 Specificity of AOB mitral cell responses Responses were recorded in Balb-c and CBA male mice. Excitatory responses (red) are most often specific for one strain and one sex of stimulus animal. Thus, firing by these neurons encodes investigation of a specific sex and strain.

27 Other types of tuning No neuron responded to all animals. However, some had broader specificity than those in the previous slide; 10.9 is excited by Balb-c (both sexes) and inhibited by CBA (both sexes). Some neurons (e.g. 4.3) responded only to castrated males of a particular strain; these were apparently perceived as distinct from either males or females.

28 Distinction between different parts of the same animal The mice investigated primarily the face (solid lines) and the anogenital regions (hatched lines) of the stimulus animals. These regions may be rich in secretions recognized in the VNO. Some neurons responded only to one of these areas. These neurons responded only to the face region of Balb-c female mice. This indicates that the face and anogenital regions produce different stimulatory pheromones.

29 Insights into VNO function from analysis of TRP2 KO mice In TRP2-/- animals, which lack the VNO transduction channel, VNO neurons do not respond to any urine stimuli, though they can still respond to KCl. These mice also do not emit ultrasonic vocalizations in response to urine. The VNO thus appears to lack pheromone responses.

30 Loss of VNO signaling eliminates aggressive responses to intruders TRP2-/- mice have remarkable behavioral alterations. Normal male mice will attack intruders introduced into their territory, especially if they are swabbed with male pheromone. This response is totally eliminated in TRP2-/- mice.

31 Both male and female aggression are eliminated in mice lacking VNO signaling (A) Measurement of three parameters of aggression (attack number, duration, latency) shows that TRP2-/- males (blue bars) completely lack aggression. Lactating female mice are aggressive toward intruders (B); this response is also eliminated in TRP2-/- mice (blue bars).

32 Territorial marking in TRP2-/- males Dominant mice spot urine around the cage floor (A), while subordinate mice do not make such urine marks, and restrict their urination to the periphery. When two control males are separated by a wire barrier, both mark in a dominant manner. When the barrier is removed, the mice fight, and the victor continues to mark dominantly, while the subordinated mouse suppresses marking, and begins urinating in large pools in the cage periphery (B). TRP2-/- mice do not fight, and both continue to mark in a dominant pattern (C). When a control mouse is paired with a TRP2-/- mouse, the control always wins, and the TRP2-/- mouse becomes subordinate.

33 TRP2-/- males attempt to copulate indiscriminately with both sexes The VNO was thought to be required for mating, and earlier lesion studies suggested that lesioned males would not mate. However, TRP2-/- males mate normally with females. Remarkably, though, they also mount males, which control mice never do.

34 Implications of the TRP2-/- phenotype The TRP2-/- phenotype suggests that the default pathway in the absence of VNO input is to mate with everything. VNO input causes male mice to fight rather than attempt to mate. The establishment of proper relationships among males (dominance/submission and elimination of mating attempts) apparently requires that they fight with each other. TRP2 is also required for appropriate female behavior.

35 TRP2-/- or VNO-ablated females act like males KO (orange bars) and VNOx (gray area) female mice attempt to mount male mice introduced into the cage, which normal females never do. The data imply that pheromone signals induce female behavior, and mask a latent male behavior program present in the female brain.

36 Phenotypes caused by loss of V1R or V2R responses Two other KO mice allow examination of the effects of removing signaling by only one of the two VNO neuron populations. These are the G αi2 and the β2m KOs. Loss of G αi2 eliminates signaling by the V1R neurons. β2m is a subunit of MHC proteins; its loss eliminates expression of MHCrelated V2R chaperones and therefore blocks V2R trafficking In both of these KOs, aggression is reduced or eliminated, but male-male mating does not occur. This suggests that input from both populations is required for aggression, but input from either can suppress malemale mating.

37 Mating is controlled by the MOE TRP2-/- animals still mate, so the VNO does not seem to be necessary for mating. Also, hypothalamic GnRH neurons necessary for mating are connected to the MOE, not to the VNO. Male mice lacking the MOE transduction channel, OCNC (top), or mice with chemical MOE lesions (bottom), have severe mating defects (left, sniffing; right, mounting attempts). These results indicate that volatile compounds sensed by the MOE actually control mating.

38 Model for control of mating and aggression by the VNO and MOE Gender identification and aggression are mediated by (soluble?) VNO cues. Volatile cues sensed by the MOE are necessary for mating, and inhibit aggression toward the opposite sex.

39 MUPs and deposition of pheromones Natural pheromones are often mixtures of many substances. Organic pheromones and other odorous compounds in rodent urine are bound by lipocalins called major urinary proteins (MUPs), which are present at very high concentrations (~70 mg/ml). MUPs are totally nonvolatile, of course, but could be sensed directly by VNO neurons in liquid urine. Also, MUPs slowly release bound volatile pheromones, which can then be sensed by the VNO or the MOE.

40 MUP-scent complexes mediate complex social responses in territorial animals The fact that MUPs release compounds slowly and at different rates allows animals to time when urine marks were deposited. These can thus act as complex social signals. The timing of scent marking indicates dominance/ subordination relationships that are used by females for mate choice. The composition of scent marks indicates metabolic status, which reflects health and nutrition. Urine marks represent the minutes of meetings among territorial animals.

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42 Patterns of countermarks read by females The streak was deposited as a stimulus. The male then made many countermarks around it. Diffusion of scent from the MUPs in these countermarks then allows them to be classified by age. Females prefer to mate with males that exclusively mark their territories, if these can be found. If there are foreign scent marks in all territories, they will prefer to mate with males that have made more recent countermarks around every intruder mark. The ability of a male to countermark every intruder mark demonstrates that he can successfully drive intruders off his territory, and therefore that he represents a good choice for mating.

43 Pheromone-like effects in humans Humans may exhibit a Bruce effect (termination of pregnancy upon exposure to urine of unrelated males). It is also known that women living together often have synchronized cycles, and that cycle lengthening/shortening can be produced by application of odorless underarm compounds to the upper lip of women. Compounds from follicular-phase donors shortened cycles, and those from ovulatory-phase donors lengthened cycles. This appears to be a pheromonal effect mediated through the main olfactory system. The mouse studies discussed above indicate that mating pheromones can be sensed by the MOE rather than the VNO, so these human data are not unexpected. The existence of pheromones sensed by the MOE that could control sexual attraction is good news for the perfume industry.

44 Elephant pheromones Interestingly, one of the systems for which pheromones have been best characterized is in elephants, which have stereotyped flehmen behavior in response to urine, dung, saliva, and secretions. Flehmen delivers urine and other substances directly to the VNO duct. This was recognized thousands of years ago. Early Sanskrit writings state that: Upon smelling of their own dung and urine, let them always be producing a tickling of the palate with it.

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47 Male elephants in good condition enter musth, which causes testosterone elevation and unpredictable and dangerous behavior. Musth facilitates mating and can allow a smaller male to defeat a larger one in a fight. Musth males dribble urine and secretions, and young elephants will avoid musth males based on these secretions, while other mature elephants are induced to enter musth. This was also recognized by Sanskrit writers: And from the smell of their sweat, dung, urine, and musth-fluid other elephants are instantly excited into musth. One can use flehmen and other responses to quantitate elephant reactions to fluids and candidate compounds. This allowed identification of cyclohexanone as a musth signal from males to females; musth fluids or cyclohexanone cause nursing females to form a protective phalanx around young calves, as musth males are a potential danger.

48 Remarkably, elephant pheromonal signals are identical in many cases to insect pheromones. For both insects and elephants full pheromonal activity requires a precise blend of components. This makes sense because there are few unique speciesspecific pheromone compounds, so if pheromonal signaling was determined only by the presence or absence of a compound animals would be easily confused by the many sources of such compounds. However, if a precise blend must be sensed this will be a chemical signature specific for that species. The architecture of the AOB (mitral cells integrating input from many glomeruli receiving input from multiple receptor types) may be suited to perception of such specific blends.

49 Avoidance of inbreeding via urine recognition Discrimination between individuals through urine is demonstrated by habituation trials. A rodent loses interest in sniffing pads soaked with urine or containing other secretions from one individual of the opposite sex after a few trials, but then dishabituates if urine/secretions from an individual of a different strain are used. Dishabituation is eliminated by VNO lesions. This discrimination can be used for mating preference; rodents like to mate with genetically dissimilar individuals.

50 Mating preference and the MHC Dishabituation experiments show that a mouse or rat can discriminate between individuals of congenic strains that differ only by a few amino acids in one MHC class I gene. How can this be accomplished? T-cell receptors distinguish virally infected cells from normal cells by binding selectively to MHC-peptide complexes that contain virus peptides, and animals are tolerant to complexes of their own MHC molecules with endogenous peptides. However, mice/rats that are identical at all loci except the MHC will have the same peptide repertoire. Another piece of (controversial) data: discrimination requires bacterial flora, so germ-free mice can t be distinguished.

51 One model is that MHC class I proteins are broken down in serum to 27 kd platforms that lose bound peptides and are small enough for excretion. These would be excreted in urine and might bind to organic products of bacterial metabolism. The bound products might then differ between mice with different MHCs, because the MHC binding groove contains many polymorphic residues. Mice would then smell either organic compounds volatilizing from the MHC as the urine dries, or possibly a complex of MHC with organic compound in liquid. This has been assayed using the Bruce effect (pregnancy block in response to contact with unfamiliar urine), although these data have been questioned.

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53 Reviews *Subsystem Organization of the Mammalian Sense of Smell. S. D. Munger et al., Ann. Rev. Phys. 71, (2009). *Genetic analysis of brain circuits underlying pheromone signaling. (2006) DuLac, C., and Wagner, S., Nature Rev. Genetics 40, Complementary Roles of the Main and Accessory Olfactory Systems in Mammalian Mate Recognition. M. J. Baum and K.R. Kelliher. Ann. Rev. Phys. 71, (2009). Scent wars: the chemobiology of competitive signaling in mice. (2004) Hurst, J.L., and Beynon, R.J., Bioessays 26, *Required reading

54 Papers for student presentations 1. Driving Opposing Behaviors with Ensembles of Piriform Neurons. G. B. Choi et al., Cell 146, (2011). 2. Molecular organization of vomeronasal chemoreception. Y. Isogai et al., Nature 478, (2011). 3. A natural polymorphism alters odour and DEET sensitivity in an insect odorant receptor. M. Pellegrino et al., Nature 478, (2011).