Pamela W. Feldhoff

Pheromonal activation of vomeronasal neurons in plethodontid salamanders

Pheromones from the mental glands of male plethodontid salamanders increase sexual receptivity in conspecific females. The pheromone enters the vomeronasal organ during courtship to produce this effect. Vomeronasal neurons from female Plethodon shermani were examined following exposure to male pheromone or saline placed on the nares. Agmatine was used in conjunction with the pheromone to enable immunocytochemical visualization of chemosensory neurons that were activated by the pheromone. Olfactory neurons exposed to pheromone or saline, and vomeronasal neurons exposed to saline did not demonstrate significant labeling. A population of vomeronasal neurons was intensely labeled following exposure to the pheromone. This study suggests that a specific population of vomeronasal neurons in a female plethodontid salamander is responsible for transmitting pheromonal information to the brain to produce modifications in behavior.

A new vertebrate courtship pheromone, PMF, affects female receptivity in a terrestrial salamander

Vertebrate pheromones that affect female receptivity have been documented only in salamanders. These courtship pheromones have been investigated most intensively in plethodontid salamanders. The source of the plethodontid courtship pheromone is the male’s submandibular (mental) gland, which produces a multiprotein secretion. In earlier work with our main study species, Plethodon shermani (the red-legged salamander), an extract of protein secretions obtained from male mental glands acted to increase sexual receptivity in females. In addition, one particular protein in the gland secretion, plethodontid receptivity factor (PRF), could act alone to increase female receptivity. We now report that a second protein, termed ‘plethodontid modulating factor’ (PMF), acts oppositely to reduce female receptivity. The natural courtship pheromone blend thus includes two proteins that separately produce opposing messages, even though the combined effect of both proteins is to increase female receptivity.

The administration of cobra venom factor reduces post-ischemic cerebral injury in adult and neonatal rats

The role of complement in post-ischemic cerebral injury is incompletely understood. Therefore, experiments were designed to test the effect of complement depletion on cerebral infarct volume in adult rats and cerebral atrophy in neonatal rats. Cerebral infarcts were induced in adult rats by transient filamentous occlusion of the right middle cerebral artery (MCAO). Cerebral atrophy was induced by subjecting 7-day-old rats to ligation of the right common carotid artery followed by 2.5h of hypoxia (8% O2). Forty-eight hours after MCAO, coronal sections of adult brains were obtained and stained with 2,3,5-triphenyl tetrazolium chloride. The infant rat brains were removed for analysis 6 weeks after the hypoxic-ischemic insult. Volumes of infarcts and normal hemispheric parenchyma were quantified by computer-based planimetry. Twenty-four hours prior to MCAO (adults) or hypoxia-ischemia (neonates), each animal received an i.p. injection of either 1 mcg/g body weight cobra venom factor (CVF; adult n=11; neonatal n=20) or normal saline (adult n=12; neonatal n=24). In the neonates, a second dose of CVF or saline was administered 2 days after hypoxia-ischemia. The administration of CVF significantly reduced: (1) post-ischemic cerebral infarct volume in the adults and (2) post-hypoxic-ischemic cerebral atrophy in the neonates. Therefore, complement activation augmented post-ischemic cerebral injury in adult and neonatal rats. Complement depletion induced by CVF significantly reduced post-ischemic cerebral infarct volume and atrophy in adult and neonatal rats.

Male pheromone protein components activate female vomeronasal neurons in the salamander Plethodon shermani

BackgroundThe mental gland pheromone of male Plethodon salamanders contains two main protein components: a 22 kDa protein named Plethodon Receptivity Factor (PRF) and a 7 kDa protein named Plethodon Modulating Factor (PMF), respectively. Each protein component individually has opposing effects on female courtship behavior, with PRF shortening and PMF lengthening courtship. In this study, we test the hypothesis that PRF or PMF individually activate vomeronasal neurons. The agmatine-uptake technique was used to visualize chemosensory neurons that were activated by each protein component individually.ResultsVomeronasal neurons exposed to agmatine in saline did not demonstrate significant labeling. However, a population of vomeronasal neurons was labeled following exposure to either PRF or PMF. When expressed as a percent of control level labeled cells, PRF labeled more neurons than did PMF. These percentages for PRF and PMF, added together, parallel the percentage of labeled vomeronasal neurons when females are exposed to the whole pheromone.ConclusionThis study suggests that two specific populations of female vomeronasal neurons are responsible for responding to each of the two components of the male pheromone mixture. These two neural populations, therefore, could express different receptors which, in turn, transmit different information to the brain, thus accounting for the different female behavior elicited by each pheromone component.

Plethodontid modulating factor, a hypervariable salamander courtship pheromone in the three-finger protein superfamily

The soluble members of the three‐finger protein superfamily all share a relatively simple ‘three‐finger’ structure, yet perform radically different functions. Plethodontid modulating factor (PMF), a pheromone protein produced by the lungless salamander, Plethodon shermani, is a new and unusual member of this group. It affects female receptivity when delivered to the females nares during courtship. As with other plethodontid pheromone genes, PMF is hyperexpressed in a specialized male mental (chin) gland. Unlike other plethodontid pheromone genes, however, PMF is also expressed at low levels in the skin, liver, intestine and kidneys of both sexes. The PMF sequences obtained from all tissue types were highly variable, with 103 unique haplotypes identified which averaged 35% sequence dissimilarity (range 1–60%) at the protein level. Despite this variation, however, all PMF sequences contained a conserved ≈ 20‐amino‐acid secretion signal sequence and a pattern of eight cysteines that is also found in cytotoxins and short neurotoxins from snake venoms, as well as xenoxins from Xenopus. Although they share a common cysteine pattern, PMF isoforms differ from other three‐finger proteins in: (a) amino‐acid composition outside of the conserved motif; (b) length of the three distinguishing ‘fingers’; (c) net charge at neutral pH. Whereas most three‐finger proteins have a net positive charge at pH 7.0, PMF has a high net negative charge at neutral pH (pI range of most PMFs 3.5–4.0). Sequence comparisons suggest that PMF belongs to a distinct multigene subfamily within the three‐finger protein superfamily.

A Recombinant Courtship Pheromone Affects Sexual Receptivity in a Plethodontid Salamander

Pheromones are important chemical signals for many vertebrates, particularly during reproductive interactions. In the terrestrial salamander Plethodon shermani, a male delivers proteinaceous pheromones to the female as part of their ritualistic courtship behavior. These pheromones increase the females receptivity to mating, as shown by a reduction in courtship duration. One pheromone component in particular is plethodontid receptivity factor (PRF), a 22-kDa protein with multiple isoforms. This protein alone can act as a courtship pheromone that causes the female to be more receptive. We used a bacterial expression system to synthesize a single recombinant isoform of PRF. The recombinant protein was identical to the native PRF, based on mass spectrometry, circular dichroism spectra, and a behavioral bioassay that tested the effects of recombinant PRF (rPRF) on female receptivity (21% reduction in courtship duration). The rPRF appears to mimic the activity of a mixture of PRF isoforms, as well as a mixture of multiple different proteins that comprise the male courtship gland extract. Pheromones that are peptides have been characterized for some vertebrates; to date, however, rPRF is one of only 2 synthesized vertebrate proteins to retain full biological activity.

Courtship pheromone–induced c-Fos-like immunolabeling in the female salamander brain

Plethodontid salamanders display intricate courtship behaviors. Proteinaceous courtship pheromones were recently discovered in the submandibular (mental) gland of the male Plethodon shermani, the red-legged salamander. Behavioral studies showed that these male pheromones are delivered by direct contact to the female snout and modulate her receptivity during courtship. Previous reports demonstrated that experimental application of courtship pheromones activates vomeronasal sensory neurons in P. shermani. The present study investigated the CNS response to courtship pheromones in that species using immunocytochemical detection of the immediate-early gene product c-Fos. The results show that application of a male gland extract to females activated Fos-like immunolabeling in the extended vomeronasal amygdala of the accessory olfactory system, as well as in the preoptic area and ventromedial hypothalamus; regions of the brain known to mediate reproductive responses in vertebrates. The gland extract additionally activated Fos-like labeling in the raphe median, possibly indicating a serotonergic activation. Application of individual purified courtship pheromone proteins resulted in increases in Fos-like labeling in some of the regions activated by the complete submandibular gland extract, but the pattern of labeling was not as clear as that of the complete extract. Unlike other known vertebrate reproductive pheromones, courtship pheromones in P. shermani were effective only at a high concentration. This could result from the particular mode of pheromone transfer in that species, which involves sustained direct contact between male and female. It is concluded that salamander courtship pheromones exert their influence on behavior through the vomeronasal pathway and its direct projections to the preoptic and hypothalamic regions.

A Candidate Vertebrate Pheromone, SPF, Increases Female Receptivity in a Salamander

Plethodontid (lungless) salamanders have evolved an unusual pheromone delivery system in which the male courtship pheromone is applied to the skin of the female, apparently diffusing through the mucosal-rich epithelia into her superficial capillary system. In Desmognathus ocoee, a plethodontid salamander that uses the diffusion mode of pheromone delivery, we conducted a behavioural bioassay to test a 20–25 kDa molecular weight fraction of the male courtship pheromone: this fraction was effective in increasing female receptivity. The principal component of the D. ocoee pheromone fraction was identified as a 25 kDa protein that had significant sequence similarity with the precursor of a newt reproductive pheromone (a decapeptide termed sodefrin). We termed the principal protein component in the D. ocoee pheromone “Sodefrin Precursor-like Factor” (SPF). SPF also occurs in other plethodontid salamanders, including species of Plethodon, Aneides and Eurycea. Across these species, SPF is a highly variable protein that bears the signature of positive selection. The presence of SPF in distantly related genera suggests that the sodefrin precursor gene has been retained as a courtship signal throughout the evolutionary radiation of plethodontid salamanders.

Effects of androgens on behavioral and vomeronasal responses to chemosensory cues in male terrestrial salamanders (Plethodon shermani)

Chemosensory stimuli and sex steroid hormones are both required for the full expression of social behaviors in many species. The terrestrial salamander, Plethodon shermani, is an emerging nonmammalian system for investigating the nature and evolution of pheromonal communication, yet little is known regarding the role of sex steroid hormones. We hypothesized that increased circulating androgen levels in male P. shermani enhance chemoreception through morphological, behavioral, and physiological mechanisms. Experimental elevation of plasma androgens increased development of cirri, morphological structures thought to enhance the transfer of chemosensory cues from the substrate to the vomeronasal organ (VNO). Elevated plasma androgens also increased expression of a chemo-investigatory behavior (nose tapping) and increased preference for some female-derived chemosensory cues. Male-produced courtship pheromones activated a large number of cells in the VNO as measured by the method of agmatine uptake. However, androgen levels did not affect the total number of vomeronasal cells activated by male-produced courtship pheromones. Future studies will determine whether androgens potentially modulate responsiveness of the VNO to female-derived (as opposed to male-derived) chemosensory cues.

Exposure to pheromones increases plasma corticosterone concentrations in a terrestrial salamander.

Sensory cues involved in social interactions can influence plasma steroid hormone concentrations. Although pheromonal communication is common in amphibians, it is unknown whether pheromones can alter hormone levels in amphibians as they do in mammals. We tested whether courtship pheromones would alter steroid hormone concentrations in male and female terrestrial salamanders (Plethodon shermani). Plasma corticosterone concentrations were elevated in male salamanders exposed to mental gland courtship pheromones, as compared to males exposed to female skin secretions or a saline control. Chemosensory cues had no effect on testosterone levels in males or on corticosterone or estradiol levels in females. These results provide the first evidence that pheromones have priming effects on the endocrine system in amphibians.