Presence of sodefrin precursor-like factor pheromone candidates in mental and dorsal tail base glands in the plethodontid salamander, Karsenia koreana.

Plethodontid salamanders are well known for their distinct courtship rituals and the associated pheromonal signaling. However, little is known about pheromones produced in the lone Asian plethodontid species Karsenia koreana. Here, we examined the localization patterns of proteins of the sodefrin precursor-like factor (SPF) pheromone system in K. koreana. Using an antibody generated against SPF proteins from another plethodontid, Desmognathus ocoee, we tested three types of skin glands in K. koreana males via immunohistochemistry: the mental gland and two types of dorsal tail base glands-caudal courtship glands and dorsal granular glands. SPF immunoreactivity was detected in the known courtship gland, the mental gland, as well as granular glands, but not in caudal courtship glands. Due to immunoreaction specificity, we hypothesize the proteins of the SPF system in K. koreana and D. ocoee are structurally and functionally related and are used as courtship pheromones in K. koreana. Also, we hypothesize that K. koreana males transmit SPF to the female during the tail-straddling walk via dorsal granular glands. Finally, K. koreana male caudal courtship glands may be producing SPF proteins that are not recognized by our SPF antibody or these glands may play a different role in courtship than anticipated.

Recurrent Co-Option and Recombination of Cytokine and Three Finger Proteins in Multiple Reproductive Tissues Throughout Salamander Evolution.

Reproductive proteins evolve at unparalleled rates, resulting in tremendous diversity of both molecular composition and biochemical function between gametes of different taxonomic clades. To date, the proteomic composition of amphibian gametes is largely a molecular mystery, particularly for Urodeles (salamanders and newts) for which few genomic-scale resources exist. In this study, we provide the first detailed molecular characterization of gametes from two salamander species (Plethodon shermani and Desmognathus ocoee) that are models of reproductive behavior. Long-read PacBio transcriptome sequencing of testis and ovary of both species revealed sex-specific expression of many genes common to vertebrate gametes, including a similar expression profile to the egg coat genes of Xenopus oocytes. In contrast to broad conservation of oocyte genes, major testis transcripts included paralogs of salamander-specific courtship pheromones (PRF, PMF, and SPF) that were confirmed as major sperm proteins by mass spectrometry proteomics. Sperm-specific paralogs of PMF and SPF are likely the most abundant secreted proteins in P. shermani and D. ocoee, respectively. In contrast, sperm PRF lacks a signal peptide and may be expressed in cytoplasm. PRF pheromone genes evolved independently multiple times by repeated gene duplication of sperm PRF genes with signal peptides recovered through recombination with PMF genes. Phylogenetic analysis of courtship pheromones and their sperm paralogs support that each protein family evolved for these two reproductive contexts at distinct evolutionary time points between 17 and 360 million years ago. Our combined phylogenetic, transcriptomic and proteomic analyses of plethodontid reproductive tissues support that the recurrent co-option and recombination of TFPs and cytokine-like proteins have been a novel driving force throughout salamander evolution and reproduction.

An annual cycle of gene regulation in the red-legged salamander mental gland: from hypertrophy to expression of rapidly evolving pheromones.

BACKGROUND: Cell differentiation is mediated by synchronized waves of coordinated expression for hundreds to thousands of genes, and must be regulated to produce complex tissues and phenotypes. For many animal species, sexual selection has driven the development of elaborate male ornaments, requiring sex-specific differentiation pathways. One such male ornament is the pheromone-producing mental gland of the red-legged salamander (Plethodon shermani). Mental gland development follows an annual cycle of extreme hypertrophy, production of pheromones for the ~ 2 month mating season, and then complete resorption before repeating the process in the following year. At the peak of the mating season, the transcriptional and translational machinery of the mental gland are almost exclusively redirected to the synthesis of rapidly evolving pheromones. Of these pheromones, Plethodontid Modulating Factor (PMF) has experienced an unusual history: following gene duplication, the protein coding sequence diversified from positive sexual selection while the untranslated regions have been conserved by purifying selection. The molecular underpinnings that bridge the processes of gland hypertrophy, pheromone synthesis, and conservation of the untranslated regions remain to be determined. RESULTS: Using Illumina sequencing, we prepared a de novo transcriptome of the mental gland at six stages of development. Differential expression analysis and immunohistochemistry revealed that the mental gland initially adopts a highly proliferative, almost tumor-like phenotype, followed by a rapid increase in pheromone mRNA and protein. One likely player in this transition is Cold Inducible RNA Binding Protein (CIRBP), which selectively and cooperatively binds the highly conserved PMF 3' UTR. CIRBP, along with other proteins associated with stress response, have seemingly been co-opted to aid in mental gland development by helping to regulate pheromone synthesis. CONCLUSIONS: The P. shermani mental gland utilizes a complex system of transcriptional and post-transcriptional gene regulation to facilitate its hypertrophication and pheromone synthesis. The data support the evolutionary interplay of coding and noncoding segments in rapid gene evolution, and necessitate the study of co-evolution between pheromone gene products and their transcriptional/translational regulators. Additionally, the mental gland could be a powerful emerging model of regulated tissue proliferation and subsequent resorption within the dermis and share molecular links to skin cancer biology.

Olfactory effects of a hypervariable multicomponent pheromone in the red-legged salamander, Plethodon shermani.

Chemical communication via chemosensory signaling is an essential process for promoting and modifying reproductive behavior in many species. During courtship in plethodontid salamanders, males deliver a mixture of non-volatile proteinaceous pheromones that activate chemosensory neurons in the vomeronasal epithelium (VNE) and increase female receptivity. One component of this mixture, Plethodontid Modulating Factor (PMF), is a hypervariable pheromone expressed as more than 30 unique isoforms that differ between individual males-likely driven by co-evolution with female receptors to promote gene duplication and positive selection of the PMF gene complex. Courtship trials with females receiving different PMF isoform mixtures had variable effects on female mating receptivity, with only the most complex mixtures increasing receptivity, such that we believe that sufficient isoform diversity allows males to improve their reproductive success with any female in the mating population. The aim of this study was to test the effects of isoform variability on VNE neuron activation using the agmatine uptake assay. All isoform mixtures activated a similar number of neurons (>200% over background) except for a single purified PMF isoform (+17%). These data further support the hypothesis that PMF isoforms act synergistically in order to regulate female receptivity, and different putative mechanisms are discussed.

Co-option and evolution of non-olfactory proteinaceous pheromones in a terrestrial lungless salamander.

Gene co-option is a major force in the evolution of novel biological functions. In plethodontid salamanders, males deliver proteinaceous courtship pheromones to the female olfactory system or transdermally to the bloodstream. Molecular studies identified three families of highly duplicated, rapidly evolving pheromones (PRF, PMF, and SPF). Analyses for Plethodon salamanders revealed pheromone mixtures of primarily PRF and PMF. The current study demonstrates that in Desmognathus ocoee--a plesiomorphic species with transdermal delivery--SPF is the major pheromone component representing >30% of total protein. Chromatographic profiles of D. ocoee pheromones were consistent from May through October. LC/MS-MS analysis suggested uniform SPF isoform expression between individual male D. ocoee. A gene ancestry for SPF with the Three-Finger Protein superfamily was supported by intron-exon boundaries, but not by the disulfide bonding pattern. Further analysis of the pheromone mixture revealed paralogs to peptide hormones that contained mutations in receptor binding regions, such that these novel molecules may alter female physiology by acting as hormone agonists/antagonists. Cumulatively, gene co-option, duplication, and neofunctionalization have permitted recruitment of additional gene families for pheromone activity. Such independent co-option events may be playing a key role in salamander speciation by altering male traits that influence reproductive success.

Proteomic analyses of courtship pheromones in the redback salamander, Plethodon cinereus.

The evolutionary success of plethodontid salamanders for ~100 MY is due partly to the use of courtship pheromones that regulate female receptivity. In ~90 % of plethodontid species, males deliver pheromones by "scratching" a female's dorsum, where pheromones diffuse transdermally into the bloodstream. However, in a single clade, representing ~10 % of Plethodon spp., males apply pheromones to the female's nares for olfactory delivery. Molecular studies have identified three major pheromone families: Plethodontid Receptivity Factor (PRF), Plethodontid Modulating Factor (PMF), and Sodefrin Precursor-like Factor (SPF). SPF and PMF genes are relatively ancient and found in all plethodontid species; however, PRF is found exclusively in the genus Plethodon - which includes species with transdermal, olfactory, and intermediate delivery behaviors. While previous proteomic analyses suggested PRF and PMF are dominant in slapping species and SPF is dominant in non-Plethodon scratching species, it was unclear how protein expression of different pheromone components may vary across delivery modes within Plethodon. Therefore, the aim of this study was to proteomically characterize the pheromones of a key scratching species in this evolutionary transition, Plethodon cinereus. Using mass spectrometry-based techniques, our data support the functional replacement of SPF by PRF in Plethodon spp. and an increase in PMF gene duplication events in both lineage-dependent and delivery-dependent manners. Novel glycosylation was observed on P. cinereus PRFs, which may modulate the metabolism and/or mechanism of action for PRF in scratching species. Cumulatively, these molecular data suggest that the replacement of pheromone components (e.g., SPF by PRF) preceded the evolutionary transition of the functional complex from transdermal to olfactory delivery.

Structural insights into the evolution of a sexy protein: novel topology and restricted backbone flexibility in a hypervariable pheromone from the red-legged salamander, Plethodon shermani.

In response to pervasive sexual selection, protein sex pheromones often display rapid mutation and accelerated evolution of corresponding gene sequences. For proteins, the general dogma is that structure is maintained even as sequence or function may rapidly change. This phenomenon is well exemplified by the three-finger protein (TFP) superfamily: a diverse class of vertebrate proteins co-opted for many biological functions - such as components of snake venoms, regulators of the complement system, and coordinators of amphibian limb regeneration. All of the >200 structurally characterized TFPs adopt the namesake "three-finger" topology. In male red-legged salamanders, the TFP pheromone Plethodontid Modulating Factor (PMF) is a hypervariable protein such that, through extensive gene duplication and pervasive sexual selection, individual male salamanders express more than 30 unique isoforms. However, it remained unclear how this accelerated evolution affected the protein structure of PMF. Using LC/MS-MS and multidimensional NMR, we report the 3D structure of the most abundant PMF isoform, PMF-G. The high resolution structural ensemble revealed a highly modified TFP structure, including a unique disulfide bonding pattern and loss of secondary structure, that define a novel protein topology with greater backbone flexibility in the third peptide finger. Sequence comparison, models of molecular evolution, and homology modeling together support that this flexible third finger is the most rapidly evolving segment of PMF. Combined with PMF sequence hypervariability, this structural flexibility may enhance the plasticity of PMF as a chemical signal by permitting potentially thousands of structural conformers. We propose that the flexible third finger plays a critical role in PMF:receptor interactions. As female receptors co-evolve, this flexibility may allow PMF to still bind its receptor(s) without the immediate need for complementary mutations. Consequently, this unique adaptation may establish new paradigms for how receptor:ligand pairs co-evolve, in particular with respect to sexual conflict.

Proteomic and UTR analyses of a rapidly evolving hypervariable family of vertebrate pheromones.

During the annual mating season, the mental gland of male plethodontid salamanders diverts its protein synthesizing capacity to the production of courtship pheromones that increase female receptivity. Plethodontid modulating factor (PMF), a highly disulfide-bonded 7-kDa pheromone, shows unusual hypervariability with each male expressing >30 isoforms. Twenty-eight PMFs were purified and matched by proteomic analyses to cDNA sequences. In contrast to coding sequence hypervariability, the untranslated regions (UTRs) show extraordinary conservation, no predicted microRNA binding sites, and an overlapping triplet polyadenylation signal. Full-length cDNA sequencing revealed three PMF gene classes containing subclasses of clustered sequences that support ≥ 13 PMF gene duplications. The unusual phenomena of hypervariable coding regions embedded within extremely conserved UTRs is proposed to occur by a disjunctive evolutionary process. During the short courtship season, the UTRs are hypothesized to subsume and coordinate the transcriptional and translational regulatory mechanisms of the mental gland. PMF, as a secreted protein with limited metabolic feedback in the male, is under minimal mutational restraint and thus has experienced highly accelerated rates of evolution. Consequently, plethodontid salamanders may provide a unique model for furthering our understanding of the selective forces that determine differential rates of gene duplication and evolution in protein families.

Elevated plasma corticosterone increases metabolic rate in a terrestrial salamander.

Plasma glucocorticoid hormones (GCs) increase intermediary metabolism, which may be reflected in whole-animal metabolic rate. Studies in fish, birds, and reptiles have shown that GCs may alter whole-animal energy expenditure, but results are conflicting and often involve GC levels that are not physiologically relevant. A previous study in red-legged salamanders found that male courtship pheromone increased plasma corticosterone (CORT; the primary GC in amphibians) concentrations in males, which could elevate metabolic processes to sustain courtship behaviors. To understand the possible metabolic effect of elevated plasma CORT, we measured the effects of male courtship pheromone and exogenous application of CORT on oxygen consumption in male red-legged salamanders (Plethodon shermani). Exogenous application of CORT elevated plasma CORT to physiologically relevant levels. Compared to treatment with male courtship pheromone and vehicle, treatment with CORT increased oxygen consumption rates for several hours after treatment, resulting in 12% more oxygen consumed (equivalent to 0.33 J) during our first 2h sampling period. Contrary to our previous work, treatment with pheromone did not increase plasma CORT, perhaps because subjects used in this study were not in breeding condition. Pheromone application did not affect respiration rates. Our study is one of the few to evaluate the influence of physiologically relevant elevations in CORT on whole-animal metabolism in vertebrates, and the first to show that elevated plasma CORT increases metabolism in an amphibian.

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.

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 female's 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.

The effects of sex on chemosensory communication in a terrestrial salamander (Plethodon shermani).

Although much evidence reveals sexually dimorphic processing of chemosensory cues by the brain, potential sex differences at more peripheral levels of chemoreception are understudied. In plethodontid salamanders, the volume of the vomeronasal organ (VNO) is almost twice as large in males as compared to females, both in absolute and relative size. To determine whether the structural sexual dimorphism in VNO volume is associated with sex differences in other peripheral aspects of chemosensation, we measured sex differences in chemo-investigation and in responsiveness of the VNO to chemosensory cues. Males and females differed in traits influencing stimulus access to VNO chemosensory neurons. Males chemo-investigated ("nose tapped") neutral substrates and substrates moistened with female body rinses more than did females. Compared to females, males had larger narial structures (cirri) associated with the transfer of substrate-borne chemical cues to the lumen of the VNO. These sex differences in chemo-investigation and narial morphology likely represent important mechanisms for regulating sex differences in chemical communication. In contrast, males and females did not differ in responsiveness of VNO chemosensory neurons to male mental gland extract or female skin secretions. This important result indicates that although males have a substantially larger VNO compared to females, the male VNO was not more responsive to every chemosensory cue that is detected by the VNO. Future studies will determine whether the male VNO is specialized to detect a subset of chemosensory cues, such as female body rinses or female scent marks.

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 female's 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 approximately 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.

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.

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

BACKGROUND: The 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. RESULTS: Vomeronasal 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. CONCLUSION: This 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.

Recombinant rat IL-1beta and IL-6 synergistically enhance C3 mRNA levels and complement component C3 secretion by H-35 rat hepatoma cells.

Hepatic synthesis of complement component C3 is regulated in part by inflammatory cytokines. Rat models are frequently employed to investigate pathogenic roles of complement and cytokines. However, cytokines obtained from species other than the rat were used in previous studies of cytokine regulation of C3 synthesis in rat hepatocytes or hepatoma cells. It is not known whether these prior reports predict hepatocellular responses evoked by rat cytokines. Therefore, H-35 rat hepatoma cells were employed to measure the effect of recombinant rat IL-1beta, IL-6, IFN-gamma, and TNF-alpha on C3 protein secretion and C3 mRNA levels quantified by ELISA and quantitative RT-PCR. Compared to untreated control cells, H-35 cells treated with IL-1beta, IL-6, and IFN-gamma increased C3 secretion approximately 10-, 4-, and 2-fold, respectively. TNF-alpha was toxic, precluding further analysis. IL-1beta and IL-6 demonstrated synergy with respect to the quantity and rate of increase of C3 mRNA measured and the magnitude of C3 protein secretion. Previous reports using non-rat cytokines did not consistently predict H-35 responses to rat cytokines. Consequently, we recommend the use of rat cytokines in rat models that include analysis of cytokine-mediated events.

Stabilizing selection on behavior and morphology masks positive selection on the signal in a salamander pheromone signaling complex.

Natural selection maintains the integration and coordination of sets of phenotypic characters that collectively perform a task. In functional complexes in which characters span molecular to behavioral levels of organization, we might then expect similar modes of selection to produce similar patterns in evolutionary divergence at each level. To test this expectation, we diagnosed selection at behavioral, morphological, and molecular levels for courtship pheromone signaling by plethodontid salamanders. At the levels of morphology and behavior tens of millions of years of stasis (stabilizing selection) occur on each side of a transition from vaccination to olfactory delivery modes. As a proxy for the molecular level, we used plethodontid receptivity factor (PRF), a protein that is an active component of the pheromone. We cloned PRF from 12 Plethodon spp. spanning the delivery transition and obtained multiple alleles from each individual surveyed. Analyses of 61 alleles for PRF identified elevated nonsynonymous over synonymous substitution rates along lineages in a molecular phylogeny, and at 8% of sites in the protein, indicating that positive (directional) selection has acted on this vertebrate pheromone gene. Structural models showed PRF is in a family of cytokines characterized by a four-alpha-helix bundle. Positive selection in PRF was associated with receptor binding sites that are under purifying selection in other cytokines of that family. The evolutionary dynamics of the plethodontid pheromone delivery complex consists of stabilizing selection on morphological and behavioral aspects of signal delivery but positive selection on the signal mediated by receptors. Thus, different selection modes prevail at different levels in this reproductive functional complex. Evolutionary studies of integrated sets of characters therefore require separate analyses of selective action at each level.

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.

The administration of complement component C9 augments post-ischemic cerebral infarction volume in neonatal rats.

To determine whether ischemic cerebral infarction is mediated in part by complement component C9, C9-deficient neonatal rats were subjected to unilateral cerebral ischemia. Brains were harvested 24 h later, stained with 2,3,5-triphenyl tetrazolium chloride, and cerebral infarct volumes were quantified by computer-based planimetry. Compared with buffer, prophylactic intraperitoneal (i.p.) administration of the complement inhibitors soluble complement receptor type 1 (sCR1), a molecular hybrid of sCR1 and the selectin inhibitor sialyl Lewis x (sCR1-sLex), or cobra venom factor did not affect the cerebral infarct volume. In contrast, i.p. human C9 (75 microg/g body weight) significantly increased the volume of infarct located 6 through 10 mm posterior to the frontal pole. Therefore, in the post-ischemic brain, C9 was neurotoxic and augmented the focal cerebral infarct volume.