BACKGROUND
understanding the determinants that influence social behavior of animals has been a matter of numerous investigations, not least because of the potential to gain insight into human abnormal behavior.
both genetic and environmental factors, including epigenetics and learning experiences or cultural conditioning, shape complex behaviors and social interactions  <cit> .
several genes have been identified that regulate social behavior of mice and other organisms  <cit>  and current research is focused on uncovering how these genes affect the molecular and neural mechanisms that underlie these behaviors.
social interactions and the display of social behavior require recognition of an interaction partner.
mice, like most vertebrates, use olfactory cues  to identify other individuals of their own and other species.
the successful identification then initiates an appropriate behavioral response.
pheromones convey information about kinship, gender, sexual receptivity, social status, or food toxicity, and therefore trigger innate social behaviors, ranging from parental care for offspring or aggression towards an intruder of their territory, to reproductive behaviors.
thus, pheromones regulate most aspects of social interactions between mice .
both volatile and non-volatile substances, including proteins and large organic compounds, can act as pheromones  <cit> .
in most animals, volatile pheromones are detected by g protein-coupled receptors in the two primary olfactory sensory organs, the main olfactory epithelium , and the vomeronasal organ  located at the bottom of the nasal cavity.
the moe is thought to facilitate associative olfactory learning and instinctive behaviors, while the vno also detects non-volatile pheromones and is considered specialized for innate responses.
humans and some primates lack a functional vno and may detect pheromones in the moe .
the ability to recognize and to distinguish between familiar and unfamiliar individuals is the result of an olfactory learning process.
while not yet well understood, this process involves specific pheromone activation profiles of neurons in the vno  <cit>  and neurogenesis in the subventricular zone   <cit> , and was shown to be supported by prolactin  <cit> .
these factors contribute to the immediate recognition of an individual, an olfactory learning process, and the formation of an olfactory memory that determines future behavior .
estrogens and androgens, and the two closely related hormones oxytocin and vasopressin have been implicated in social recognition and/or social odor memory formation .
however, how pheromones are processed, once an opponent is recognized, to ultimately elicit specific behavioral responses is largely unknown.
we and others have previously characterized the small gtpase m-ras/r-ras <dig>  which is closely related to members of the p <dig> ras family of oncogenes  that are frequently mutated in many types of human cancers .
m-ras may be overexpressed in some human cancers  <cit> , but mutations overall are very rare and no activating mutations in the typical hot spots  have been found in over  <dig>  sequenced cancers .
thus, its role in human cancers is presently unclear.
initial studies of mice lacking m-ras did not reveal any obvious abnormalities  <cit> .
we have serendipitously discovered changes in social behavior of mras−/− mice.
we have described these changes and have explored some of the possible causes in the present study.
RESULTS
social and aggressive behavioral analysis of wt and mras−/− males
high levels of aggression amongst the males in our mras−/− colony, especially after cage changes, prompted us to perform a formal analysis of social and aggressive behavior.
we subjected mras−/− males to resident-intruder tests and compared their behavior to that of wt males.
in addition, we exposed resident males to intruders of the opposite genotype to determine if this would affect interactions.
we visualized the interactions by creating ‘behavior heatmaps’ of observable behaviors  displayed by each mouse over time  and quantified these behaviors .
there were striking differences between behaviors exhibited by wt and mras−/− males in their roles as residents.fig.
<dig> altered social behavior in mras
−/−  males.
a cumulative duration of aggressive behavior by the resident was scored during 10-min resident-intruder tests.
mras
−/−  residents displayed enhanced aggressive behavior towards ko and wtsw intruders but not towards wt or ob <dig> intruders .
wt/wt  vs. ko/ko, p =  <dig> ; ko/ko vs. ko/wt, p =  <dig> ; ko/ko vs. ko/ob <dig>  p =  <dig> ; ko/wt vs. ko/wtsw, p =  <dig> ; ko/ob <dig> vs. ko/wtsw, p =  <dig> ; all others not significant.
b social investigations  by the residents during the first 5 min of the resident-intruder tests were scored.
wt/wt  vs. ko/ko, p <  <dig> ; ko/ko vs. ko/ob <dig>  p =  <dig> .
c social investigations by the intruders during the first five min.
of the resident-intruder tests were scored.
there were no significant differences for both wt and ko intruders.
d digging behavior by the residents was scored during the first 5 min of the resident-intruder tests.
p =  <dig>  for wt/wt vs. ko/ko.
e digging behavior by the intruders was scored during the first 5 min of the resident-intruder tests.
there were no significant differences for both wt and ko intruders.
b–e
error bars represent sem.
f ‘corner’ behavior was scored for residents, showing more frequent anxiety-related behavior of mras
−/−  males.
p <  <dig>  for all wt residents vs. all ko residents combined; not significant between individual groups.
a–f n =  <dig> for all groups except ob <dig>  where n =  <dig>  g open field tests: the amount of time mice spent at the center of an open and brightly lit white box was scored over a period of ten min.
error bars represent sem, n =  <dig> for all groups except n =  <dig> for mras
−/− males; p =  <dig> ; females, p =  <dig> .
h mating assays: male mating behavior was scored in 10-min.
interactions between combinations of wt and ko males and females.
n =  <dig> for all groups; p =  <dig>  for wt/wt  vs. ko/ko; p =  <dig>  for ko/wt vs. ko/ko; not significant for wt/wt vs. wt/ko
when paired with intruders of their own genotype, half of the mras−/− resident males exhibited aggression .
although the c57bl/6 j mouse strain is considered low to medium aggressive  <cit> , our wt males did not exhibit aggression during the brief 10 min.
assessment period .
the difference in aggressive behavior displayed by wt vs. mras−/− residents was significant .
we also observed a significant increase in the rate of parental cannibalism of neonates in the mras−/− colony : wt breeder pairs  cannibalized  <dig> of  <dig> litters , whereas mras−/− breeder pairs  cannibalized twice as many litters .
cannibalism can be explained by parental aggression, although it can occur due to other factors, such as stress.
during resident-intruder tests with isogenic male intruders, mras−/− residents also spent significantly more time investigating intruders compared to wt residents .
in contrast, wt residents exhibited significantly increased digging behavior compared to mras−/− residents .
thus, in interactions with isogenic males, wt and mras−/− residents displayed social behaviors that differed significantly from one another.
curiously, when mras−/− resident males were paired with wt intruders, aggression decreased significantly .
mras−/− residents also spent less time investigating wt intruders less, although this did not reach significance .
in addition, mras−/− residents did not attack other knockout or transgenic mice on a c57bl/6 j background  and displayed significantly decreased social investigations towards these intruders compared to mras−/− intruders .
in contrast, wt residents did not change their behavior when paired with mras−/− intruders .
thus, the expression of aggression by mras−/− males depended on the genotype of the intruder; mras−/− residents reacted aggressively to mras−/− intruder mice but not to mice that express m-ras, while wt residents either did not make this distinction or did not react to it.
mice use pheromonal cues to identify other individuals.
thus, we evaluated the behavior of mras−/− residents in the presence of wt intruders that were swabbed with urine from mras−/− males .
wtsw intruders did not elicit aggression from wt residents , but elicited aggression from six out of ten mras−/− residents , which was similar to the frequency observed with mras−/− intruders .
there difference in aggressive behavior of mras−/− residents towards wt vs. wtsw intruders was significant .
aggressive behavior by mras−/− residents may not have been not fully restored with mras−/− social odors because attack duration was shorter on average  with wtsw intruders compared to mras−/− intruders .
wtsw intruders stimulated social investigations from mras−/− residents at levels that were similar to mras−/− intruders .
these results suggested that mras−/− males may produce urinary pheromones that are distinct in quality or quantity from wt males and that could also contain elevated levels of aggression pheromones  <cit> .
however, mras−/− pheromones alone were not sufficient to elicit aggressive behavior because wt mice were not more aggressive towards mras−/− intruders than towards wt intruders .
thus, other changes must have occurred in mras−/− males to elicit the aggressive behavioral response.
wt residents tended to spend more time investigating mras−/− intruders than wt intruders and exhibited less digging behavior in the presence of an mras−/− intruder, although these differences were not statistically significant .
in addition, sniffing and digging behavior displayed by wt intruders was irrespective of the genotype of the resident .
thus, wt males followed similar behavioral patterns with isogenic and congenic interaction partners both in their respective roles as residents and as intruders.
in contrast, mras−/− males adapted their social behaviors depending on the genotype of the interaction partner when they were residents, but not when they were intruders.
inreased anxiety-like behavior in mras−/− mice
mras−/− residents frequently retreated to the corner or short edge of the test cage during the resident-intruder test .
this ‘corner’ behavior was reminiscent of a fear response, similar to the ‘corner’ behavior observed in open field tests that has been considered anxiety-like behavior .
‘corner’ behavior was very rare with wt residents .
compared to wt mice, mras−/− mice also exhibited significantly increased anxiety-like behavior in an open field test , including significantly more time spent in the corners of the open field .
the ‘corner’ behavior by mras−/− males in the resident-intruder test was similar in the presence of intruders with different genotypes/odortypes .
this suggests that enhanced anxiety-like responses may be a general characteristic of mras−/− males that, unlike sniffing and aggressive behaviors, was not adapted to different opponents.
interactions with females
we next investigated interactions between male and female mice.
we observed a marked increase in male mounting activity among isogenic mras−/− mating pairs relative to isogenic wt mating pairs .
ten out of ten mras−/− males engaged in mating behavior within a 10 min.
test period, whereas only four of ten wt males did.
moreover, all ten mras−/− males spent more than 50 s displaying mating behavior with mras−/− females, whereas only one of ten wt males showed mounting behavior for more than 50 s with wt females .
this was likely not due to a general lack of motivation or problems exhibiting mating behavior, as wt and mras−/− males were equally effective at impregnating proestrus females when interactions were allowed to proceed over night .
to test the ability of mras−/− males to discriminate between wt and mras−/− females, females of the opposite genotype were paired with the males.
intriguingly, while wt males showed about the same amount of mounting behavior with wt and mras−/− females, mras−/− males exhibited significantly reduced mounting activity when paired with a wt female .
this result suggests that the display of increased mounting behavior by mras−/− males, like the display of aggression, may depend on the genotype of the interaction partner.
in addition, this also suggests that mras−/− females, like mras−/− males, may produce pheromones that are different from the pheromones secreted by wt females.
however, these pheromones alone were not sufficient to elicit an increase in mating behavior from wt males, and the increased mounting activity was therefore like aggression a characteristic of mras−/− males.
female mating behavior was also assessed.
we did not observe lordosis in any of the females tested , which may have been due to the short duration of the assay.
we noted that mras−/− females appeared to spent significantly more time investigating mras−/− males compared to wt females investigating wt males .
wt females also appeared to spend significantly more time investigating mras−/− males than wt males , while mras−/− females appeared to spend about the same amount of time investigating males of both genotypes .
however, female responses were likely affected by male behavior , making interpretation of female behavior difficult.
comparable levels of testosterone and sexually dimorphic genes in wt and mras−/− males
testosterone is related to aggressive behavior in animals  <cit> .
however, testosterone levels were comparable in the serum and testes of wt and mras−/− males, and those mras−/− residents that displayed territorial aggression did not exhibit significant changes in testosterone levels either .
levels of serum estradiol were also similar in wt and mras−/− males .
moreover, the expression levels of sexually dimorphic genes that have been implicated in male aggression and male and female sexual behavior  <cit>  were comparable in wt and mras−/− mice .
these findings indicated that the lack of m-ras caused changes in social behavior that depended on factors other than sex hormones, sytl <dig>  irs <dig>  cckar, or progesterone receptor.
they also indicated that mras−/− mice may secrete pheromones that differ from those of wt mice, and the possibility that mras−/− males may have an altered ability to sense pheromones and/or respond appropriately to pheromones.fig.
<dig> testosterone levels.
a serum testosterone levels in wt and mras
−/− males .
b total testosterone per mg testis tissue in wt and mras
−/− males .
c serum testosterone in aggressive  and non-aggressive  mras
−/− males.
all data is from six month-old males and shown with sd.
a–c there were no significant differences in testosterone levels between wt and mras
−/− samples
lack of social odor discrimination by mras−/− mice
mice spend more time investigating unfamiliar scents over familiar ones  <cit>  and are able to distinguish between very subtle differences in pheromone compositions amongst genetically identical mice  <cit> .
we determined whether wt and mras−/− mice would discriminate between wt and mras−/− male urinary pheromones in the absence of the owner of the scent.
as expected, wt females spent significantly greater time and frequency investigating the unfamiliar mras−/− male urine spots relative to wt urine spots .
similarly, wt males tended to spend more time investigating mras−/− urine spots, although this did not reach statistical significance .
in contrast, both mras−/− males and females spent about the same amount of time investigating wt and mras−/− male urine spots, with mras−/− males exhibiting a slight but significant preference for mras−/− urine spots .
we also tested the ability of wt and mras−/− fathers to recognize their own adult offspring.
while wt fathers, as predicted, spent significantly more time investigating the alien isogenic offspring than their own offspring , mras−/− fathers spent the same amount of time investigating both their own and the alien isogenic offspring .fig.
<dig> evidence for defective vomeronasal function in mras
−/− mice.
a urine spot investigations.
wt mice, but not mras
−/− mice, spent more time investigating an unfamiliar  over a familiar  urine spot  and visited the unfamiliar spot more frequently .
data is shown with sem; n =  <dig> for males, n =  <dig> for females; one-sample t-tests: wt females: p =  <dig>  , p =  <dig>  ; mras
−/− males: p =  <dig>  , p =  <dig>  ; all others, not significant.
b parental offspring recognition test.
mras
−/− fathers, in contrast to wt fathers, were unable to discriminate between their own offspring and an unrelated isogenic mouse.
one-sample t-tests: wt: p =  <dig> , n = 7; mras
−/−: not significant, n = 8
these results alluded to two possible underlying problems in mras−/− mice.
that they were unable to recognize familiar pheromones or to discriminate between pheromones from isogenic and congenic mice suggests that pheromone detection, possibly by the vomeronasal organ , may be compromised in mras−/− mice.
however, mras−/− males clearly displayed different behaviors with wt and mras−/− interaction partners , which paradoxically indicated that pheromones were detected by mras−/− mice.
basic olfaction by the main olfactory bulb, as determined by a buried food test, and gross olfactory bulb anatomy were similar in wt and mras−/− males .
although immediate detection of pheromones may be intact, pheromone processing and subsequent olfactory learning could be defective in mras−/− mice.
this may have rendered them unable to generate appropriate interpretations and social behaviors in response to pheromones.
alternatively, mras−/− males may produce distinct urinary pheromones that evoke increased interest in mras−/− mice .
we addressed both possibilities in subsequent experiments.
known mechanisms that contribute to olfactory learning are similar in wt and mras−/− mice
we explored several processes that are known to affect olfactory learning to determine if they may underlie an apparent defect in vno function in mras−/− mice.
first, we tested acute pheromone sensing and signaling by g protein-coupled receptors in vno neurons.
exposure to alien pheromones leads to the induction of the immediate-early genes , c-fos and egr- <dig>  <cit> , and gαo function in the vno is required for aggressive behavior  <cit> ).
we exposed wt and mras−/− males to soiled bedding from either isogenic or congenic mice for 30 min.
and analyzed ieg induction in vno tissue.
exposure to either bedding source led to a significant upregulation of c-fos .
egr- <dig> expression was also induced although not significantly .
there were no significant differences in the levels of induction of c-fos and egr- <dig> between wt and mras−/− mice, which suggested that there were also no differences in g-protein coupled signaling by vomeronasal receptors leading to induction of these two key genes.
thus, acute pheromone sensing was likely intact in mras−/− mice, which supports our conclusion from the results shown above.fig.
<dig> ieg induction, expansion of neural progenitors, and regulation of th.
a, b
c-fos and egr- <dig> gene induction in the vno after exposure to alien male bedding for 30 min.
control  mice remained in their own cage, ‘iso’ and ‘con’ refers to exposure to soiled bedding from isogenic or congenic mice, respectively.
data is shown with sd, n =  <dig> for all groups except mras
−/− exposed to ‘iso’, n =  <dig>  and mras
−/− exposed to ‘con’, n = 4; no significant effect of genotype for either ieg.
c, d the average number of ns generated  and average ns sizes  at varying concentrations of egf were similar for wt and mras
−/− neural progenitors.
there were no significant differences between wt and mras
−/− samples; n =  <dig> for experiments in c, n =  <dig> for experiments in d, error bars represent sd.
e stimulation of prlr- <dig> or prlr- <dig> with prolactin resulted in weak activation of m-ras but with different kinetics; n =  <dig> for prlr- <dig> samples ; n =  <dig> for prlr- <dig> samples; error bars represent sem.
f prolactin did not affect the number of ns generated .
g prolactin slightly stimulated the proliferation of wt or mras
−/− neural progenitors  to similar levels; n =  <dig> wt ctrl, n =  <dig> wt + prl, n =  <dig> mras
−/− ctrl, n =  <dig> mras
−/− + prl; data shown with sem.
h
tyrosine hydroxylase  gene expression in the olfactory bulbs of mated and plugged females .
data is shown with sd, n =  <dig> for all groups except mras
−/−  <dig>  dpc where n = 3; no significant effect of genotype
epidermal growth factor  is the major growth factor driving the expansion of neural progenitor cells in the svz.
as m-ras is potently activated by egf  <cit>  and is highly expressed in the brain, including the svz, the rostral-migratory stream, and the olfactory bulbs , we evaluated egf responsiveness of svz neural progenitors from wt and mras−/− mice in a neurosphere  assay.
however, the number of ns generated and their expansion with egf, including long-term proliferation, was virtually identical in wt and mras−/− cells , which suggests that expansion of neural progenitors during olfactory learning is likely similar in wt and mras−/− mice.
prolactin  acts through prlr isoforms of the cytokine receptor family and so was predicted to stimulate m-ras activation  <cit> .
indeed, transcript variants prlr- <dig> and prlr- <dig> conferred weak but consistent m-ras activation with different kinetics .
prlr- <dig>  which has the shortest intracellular domain of the prlr isoforms, did not activate m-ras .
the addition of prolactin to ns assays did not affect the number of ns generated from wt or mras−/− neural progenitors , but the average size of both wt and mras−/− ns increased slightly, and to a similar extent, with addition of prolactin to the cultures , which indicated that prolactin mildly stimulated the proliferation of nsc.
interestingly, while svz tissue expressed detectable levels of all three prlr isoforms, with the longest isoform prlr- <dig> being the most abundant, we were unable to detect any prlr transcripts in cultured nsc .
thus, the proliferative stimulation likely occurred early in vitro, either before the neural progenitors downregulated prlrs, or after supporting cells died off.
nevertheless, prolactin stimulated the expansion of neural progenitor cells to similar extents in both wt and mras−/− cells.
another factor that can contribute to changes in social behavior is a change in motivation.
interest in mating and display of aggression can be considered motivation-based behaviors that are associated with reward, and both are associated with dopamine production or signaling .
tyrosine hydroxylase  is the key enzyme in dopamine biosynthesis; it can be used as an indirect measure of dopamine production  <cit> .
we analyzed the changes in expression levels of th upon mating in the olfactory bulbs of female mice.
th rna levels changed significantly , dropping early after mating  and subsequently increasing to steady-state levels or slightly above .
the changes in th expression levels were similar in wt and mras−/− mice, which suggests that dopamine metabolism was likely not significantly different between these mice.
the urinary metabolite profile of mras−/− males may be distinct from wt males
compounds that function as pheromones can be volatile or non-volatile chemicals, or can be proteins  <cit> .
to determine whether wt and mras−/− male urine differ in protein content, we tested randomly collected urine samples by sds electrophoresis and coomassie stain.
the concentration of 21 kda major urinary proteins , which contain and may bind to pheromones, was slightly higher in mras−/− males .
however, the levels of darcin , which was identified as an attractive pheromone  <cit> , were similar in wt and mras−/− males .
next, we analyzed urine samples by nmr and mass spectrometry for urinary metabolites.
several metabolites were dysregulated in mras−/− urine .
for example, glycerate was increased  <dig> -fold and succinate was decreased  <dig> -fold.
in addition, some components of lipid metabolism were detected at different levels in wt and mras−/− urine.
sixty-four percent of the phosphatidylcholines and 43 % of the lysophosphatidylcholines tested were increased at least two-fold, and 44 % of sphingomyelins were downregulated greater than two-fold in mras−/− urine compared to wt urine.
interestingly, mras−/− male urine contained twice as much trimethylamine as wt male urine.
trimethylamine has been identified as an attractive chemosignal in mouse urine  <cit> .
the metabolomics analysis was performed only once with urine samples pooled from three males each and did not include many volatile compounds .
however, the results from the resident-intruder tests may support the conclusion that the urinary metabolite/pheromone profiles likely differ between wt and mras−/− males, because mras−/− residents reacted differently to wt vs. wtsw or mras−/− intruders .fig.
<dig> major urinary proteins  and darcin.
urine was randomly collected from six wt and mras
−/− male mice each .
two µl of urine was loaded onto a 15 % sds gel per sample.
gels were stained with coomassie blue to visualize protein.
mups are observed at  <dig> kd, darcin at approximately 16- <dig> kd
table  <dig> urine metabolomics analysis
wt and mras
−/− male urine samples were analyzed by nmr and direct-injection mass spectrometry .
total number of metabolites analyzed by nmr: 65; di-ms:  <dig>  some differences in relative metabolite quantities were confirmed by gas chromatography -ms analysis: glyceric acid: + <dig> ; l-tyrosine: + <dig> ; succinic acid: − <dig> ; hippuric acid: − <dig>
in summary, while none of the known factors contributing to pheromone sensing and olfactory learning appears to be compromised in mras−/− mice, it is possible that other, unexplored mechanisms may be affected by the absence of m-ras.
moreover, it is likely that distinct pheromones produced by mras−/− males additionally affect their social behavior.
discussion
we uncovered changes in social behavior in mice lacking m-ras, and used a graphical representation of dynamic social interactions, a behavior heatmap, to visualize differences in the ways wt and mras−/− mice dynamically interact with each other.
mras−/− males exhibited higher levels of territorial aggression with other mras−/− males and spent more time than wt males to investigate an intruder; often, these prolonged investigations culminated in a physical attack.
mras−/− mice displayed less digging behavior but increased fear-related responses such as the ‘corner’ response .
in mating assays with females, mras−/− males more readily engaged in mating behavior compared to their wt counterparts.
thus, mras−/− males showed social behaviors that were strikingly different from wt males.
the underlying causes for aggressive behavior have been the subject of intense investigations, particularly in humans.
although genetics and differentially regulated brain chemistry or neuronal circuits may play a role, aggression is also strongly influenced by experience  and increases with positive reinforcement .
another determinant of aggressive behavior is the interaction partner with his unique stimulus properties/characteristics .
some mouse strains elicit higher levels of aggression than others, and hormone status and gender of the opponent can also affect aggressive responses  <cit> .
it is likely that olfactory  cues emanating from the opponents induce aggressive behaviors in resident males  <cit> .
many different knockout mouse strains are known to exhibit changes in aggression levels  <cit> .
to our knowledge, our study is the first to show that an increase in aggressive behavior depended on two factors:  the absence of mras in the resident and  the absence of a single gene in the intruder, mras , unless compensatory changes in expression levels of other genes occurred during development.
our data supports that differentially secreted pheromones by mras−/− mice may contribute to the occurrence of aggression.
if wt and mras−/− mice indeed emit different social odors, a wt intruder would be less familiar than an mras−/− intruder for an mras−/− resident male who had grown up in the presence of mras−/− but not wt social odors.
thus, the prediction was that the wt intruder should have suffered greater aggression  <cit> .
that the opposite occurred was surprising and will be further discussed below.
the increases in aggression and mounting activity in mras−/− males were unrelated to levels of testosterone or estradiol and expression levels of genes that are considered sexually dimorphic .
the serotonin system also contributes to the regulation of social interactions, including aggression, in humans and mice.
mice deficient in 5-ht1b exhibit increases in aggressive behavior  <cit> , and greater serotonin activity positively affects social interactions in humans and animals  <cit> .
an optimal level of serotonin signaling is required during brain development for normal behavior in adulthood, and anxiety is associated with reduced function  <cit> .
it will be of interest to determine if mras−/− mice exhibit changes in the serotonin system that could promote an increase in aggression and/or in fear-related behavior .
enhanced aggression and enhanced anxiety could contribute to an inability to appropriately assess contextual risk and adjust behavior accordingly.
wt and mras−/− resident males also differed in their digging behavior.
digging was a characteristic of wt but not mras−/− residents .
digging could represent an attempt to escape, perhaps in search of shelter  <cit> .
we interpreted digging during the resident-intruder test as an expression of frustration   <cit> .
thus, it is possible that wt and mras−/− males may express frustration differently.
another interesting observation emerged from our studies: mras−/− mice failed to discriminate both between highly similar, isogenic urinary pheromones , and even between pheromones from congenic and isogenic mice, where greater differences in pheromone composition would be expected .
this could be interpreted as evidence for a defect in acute pheromone processing in mras−/− mice.
the problems could be within the vno and affect immediate detection.
however, c-fos induction in the vno in response to social odors was similar in wt and mras−/− males .
alternatively, processing of pheromone information downstream of the vno may be defective.
in particular, the medial amygdala integrates social odor cues from signals from both the main and accessory olfactory systems .
although mras−/− mice did not discriminate between pheromones in the absence of the owners of these scents, they did discriminate between isogenic and congenic mice when the animal was present and available for social interactions, and exhibited enhanced aggression and mating behavior only towards isogenic partners .
this seemed paradoxical, but suggested that pheromones were detected and that acute olfactory and/or vomeronasal function was intact in mras−/− males.
there are several possible explanations.
first, independent of pheromones or their mechanisms of detection and processing, mice may be able to detect and interpret non-olfactory contextual information.
this would depend both on the perception of the resident and on the behavior of the intruder.
the intruder with his own characteristics will affect resident behavior .
for example, wt residents may detect differences in motor patterns or anxiety in mras−/− intruders and may not feel the need to attack, and mras−/− residents paired with mras−/− intruders could sense each other’s anxiety to produce changes in behavioral patterns  <cit> .
however, this is likely only a minor factor because mras−/− residents exhibited similar levels of aggression and social investigations with mras−/− intruders and wt intruders when the latter were swabbed with mras−/− urine, but they did not attack unswabbed wt intruders even though the wt intruders did not change observable behaviors whether they were swabbed with urine or not.
second, we cannot rule out that the olfactory and/or social recognition processes may be malfunctioning in mras−/− mice, as we have only addressed a subset of molecular correlates.
immediate detection of pheromones was intact in mras−/− males, as assessed by the induction of c-fos upon pheromone exposure and the differential behavioral responses with wt and mras−/− territorial intruders .
in addition, neural progenitor cells of mras−/− mice had the same capacity to respond to an egf or prolactin stimulus cells from wt mice .
there are conceivably many more cellular and biochemical players that contribute to a highly complex learning process.
for example, we have not addressed the possibility that mras−/− mice may exhibit epigenetic modifications, which could indicate that they inherited an olfactory experience from their ancestors  <cit> .
moreover, hormones such as oxytocin, vasopressin, and serotonin have been implicated in the molecular basis of social recognition.
in particular, the closely related oxytocin and vasopressin may be involved in longer-term social recognition and modulation of behavioral responses in rodents .
mice lacking oxytocin do not display an increase in anxiety and a deficiency in the vasopressin receptor, v1ar, may result in male-specific reduction in anxiety levels .
in contrast, both male and female mras−/− mice showed higher levels of anxiety.
vasopressin may play dual roles in the regulation of aggressive behavior, facilitating or inhibiting aggression depending on the specific site of release in the brain .
it will be worthwhile to examine the contributions of these hormones to the behavioral changes in mras−/− mice.
in addition, changes in other hormones and their receptors  could drive the changes in social recognition and the associated differences in behavior.
third, despite the evidence that points to a possible defect in vno function, we cannot exclude a role for pheromones, or the processing of pheromones, in mras−/− mouse behavior.
we observed that mras−/− mice may differentially produce several urinary metabolites compared to wt mice.
we are currently exploring whether this may correlate with an enlargement of urinary bladders that frequently occurs in mras−/− males and seems to worsen with age .
one metabolite that was elevated in mras−/− urine, trimethylamine, is a known pheromone  <cit> .
by virtue of its attractive properties, trimethylamine may facilitate social interactions between mice.
interestingly, high concentrations of trimethylamine can induce an aversive response in mice.
the receptor for trimethylamine is taar  <cit> ; it will be of interest to determine if taar levels are dysregulated in mras−/− mice.
the altered secretion of trimethylamine and other urinary metabolites suggest that is very likely that the pheromone profile of mras−/− mice differs from wt mice.
it was shown that mice that are deficient in oxytocin or estrogen receptor β produce urinary substances that facilitate an aversive response  <cit> .
it is possible that mras−/− mice could produce distinct pheromones, such as trimethylamine, that may be perceived as enticing to both wt and mras−/− mice and affect behavioral responses.
darcin is another urinary protein that is perceived as attractive  <cit> , although its levels were similar in wt and mras−/− male urine .
the attraction of an mras−/− male to an as yet unidentified, mras−/−-specific pheromone could cancel out the urge to explore a novel urine spot produced by a wt male when mras−/− urine is present at the same time, and could further stimulate a wt male to explore an mras−/− urine spot .
likewise, the unknown pheromone could restore exploration and attack of wt intruders by mras−/− residents when the former were swabbed with mras−/− urine , and could explain why mras−/− fathers investigate both their own and an alien offspring equally .
the results from the offspring recognition tests, where test subjects were only exposed to airborne pheromones, suggest that the unknown, attractive pheromone could be volatile.
thus, the apparent defect in vno function may be an artifact produced by an enticing pheromone secreted by mras−/− mice.
however, even though urinary metabolites and pheromones may be differentially secreted by mras−/− mice, these alone were not sufficient to evoke an aggressive response or increased mating behavior by wt males, and additional mechanisms that could be involved with the processing of olfactory information must be dysregulated in mras−/− males for aggression and interest in mating to surface more readily.
so far we have only explored the acute detection of pheromones in the vno  and the expansion of neural progenitors  and found that these were similar in wt and mras−/− mice.
however, other molecular pathways downstream of the induction of iegs, and/or as yet unknown molecular mechanisms and neural circuits that contribute to the processing of olfactory information could be affected by the absence of m-ras.
fourth, in combination with genetics and pheromones, learning will also affect behavior.
without a learning process, memory cannot be established.
interestingly, behavioral performance of neonates in an associative olfactory learning task was shown to depend on transcript levels of rasgrf <dig>  which is an activator of m-ras  <cit> .
in addition, the mras gene is upregulated upon spatial discrimination learning in rats  <cit> .
we observed that mras−/− mice show a slight delay in spatial learning in the morris water maze test, which was likely not due to an impairment in motor function.
however, they caught up after eight training sessions and the consolidation of reference memory was similar to wt mice .
if mras needs to be upregulated for an optimal hippocampus-dependent spatial learning task, it may also need to be increased for optimal olfaction- or pheromone-dependent social recognition, and the behavioral changes we have observed in mras−/− mice could be indicative of a slight learning deficit in general.
thus, mras−/− males could take longer to process pheromone information emitted by unfamiliar wt intruders and we cannot rule out that mras−/− resident males, when given more time, might attack wt intruders eventually.
likewise, mras−/− fathers could take longer to process the similar pheromones produced by their own and alien isogenic offspring, and may require more time to determine that there are differences.
alternatively, it is also possible that there may be defects in the formation of social odor memory.
the oxytocin and vasopressin systems have roles in social odor memory consolidation  <cit> , and more experiments are required to address the possibility of their involvement and to distinguish between short-term recognition  and long-term memory formation.
in addition, mras−/− pups could be fostered to wt parents in order to test whether there are problems with olfactory recognition or if there might be a component of faulty experiential learning through a parenting defect.
finally, it is possible that the observed behavioral changes in mras−/− males could have been a consequence of ontogenetic processes caused by the deletion of the mras gene, resulting in changes in expression levels of other genes.
in addition, given the similarity to other proteins of the ras family of gtpases, especially r-ras, tc <dig>  and the p <dig> ras proteins, it is conceivable that these proteins could have taken over some of the normal functions of m-ras, but could have skewed signaling pathways through slightly different effector usage  <cit> .
roles for ras proteins in social behavior have not yet been reported, although neurofibromatosis  <dig> , which inactivates ras proteins by stimulating their gtpase activities, has been implicated in social recognition in mice  <cit> .
it will be of interest to determine whether mice with deletions of other ras proteins exhibit behavioral changes; this could provide insight into possible compensatory mechanisms in mras−/− mice.
CONCLUSIONS
in summary, mras−/− males exhibited changes in social behavior that included increased territorial aggression, increased mating behavior, and fear-like responses.
these changes were likely caused by a combination of several factors.
pheromones may be differentially produced and/or differentially sensed and interpreted by mras−/− mice.
these pheromones could interfere with the correct identification of other individuals, which in turn could affect olfactory learning and result in an inability to respond appropriately to social stimuli.
however, the behavior changes in mras−/− males cannot be explained by differences in pheromones alone, as these were not sufficient to elicit aggression or enhanced mating behavior from wt males.
other mechanisms, perhaps a signal transduction pathway that is under- or overactive by the absence of m-ras must be altered to predispose mras−/− mice to display altered behavior.
as social interactions in higher organisms are highly complex this will require more detailed investigations into the molecular pathways and neural circuits that m-ras may affect.

 understanding the determinants that influence social behavior of animals has been a matter of numerous investigations, not least because of the potential to gain insight into human abnormal behavior. both genetic and environmental factors, including epigenetics and learning experiences or cultural conditioning, shape complex behaviors and social interactions. several genes have been identified that regulate social behavior of mice and other organisms and current research is focused on uncovering how these genes affect the molecular and neural mechanisms that underlie these behaviors. social interactions and the display of social behavior require recognition of an interaction partner. mice, like most vertebrates, use olfactory cues to identify other individuals of their own and other species. the successful identification then initiates an appropriate behavioral response. pheromones convey information about kinship, gender, sexual receptivity, social status, or food toxicity, and therefore trigger innate social behaviors, ranging from parental care for offspring or aggression towards an intruder of their territory, to reproductive behaviors. thus, pheromones regulate most aspects of social interactions between mice. both volatile and non-volatile substances, including proteins and large organic compounds, can act as pheromones. in most animals, volatile pheromones are detected by g protein-coupled receptors in the two primary olfactory sensory organs, the main olfactory epithelium, and the vomeronasal organ located at the bottom of the nasal cavity. the moe is thought to facilitate associative olfactory learning and instinctive behaviors, while the vno also detects non-volatile pheromones and is considered specialized for innate responses. humans and some primates lack a functional vno and may detect pheromones in the moe. the ability to recognize and to distinguish between familiar and unfamiliar individuals is the result of an olfactory learning process. while not yet well understood, this process involves specific pheromone activation profiles of neurons in the vno and neurogenesis in the subventricular zone, and was shown to be supported by prolactin. these factors contribute to the immediate recognition of an individual, an olfactory learning process, and the formation of an olfactory memory that determines future behavior. estrogens and androgens, and the two closely related hormones oxytocin and vasopressin have been implicated in social recognition and/or social odor memory formation. however, how pheromones are processed, once an opponent is recognized, to ultimately elicit specific behavioral responses is largely unknown. we and others have previously characterized the small gtpase m-ras/r-ras which is closely related to members of the p ras family of oncogenes that are frequently mutated in many types of human cancers. m-ras may be overexpressed in some human cancers, but mutations overall are very rare and no activating mutations in the typical hot spots have been found in over sequenced cancers. thus, its role in human cancers is presently unclear. initial studies of mice lacking m-ras did not reveal any obvious abnormalities. we have serendipitously discovered changes in social behavior of mras−/− mice. we have described these changes and have explored some of the possible causes in the present study.  social and aggressive behavioral analysis of wt and mras−/− males high levels of aggression amongst the males in our mras−/− colony, especially after cage changes, prompted us to perform a formal analysis of social and aggressive behavior. we subjected mras−/− males to resident-intruder tests and compared their behavior to that of wt males. in addition, we exposed resident males to intruders of the opposite genotype to determine if this would affect interactions. we visualized the interactions by creating ‘behavior heatmaps’ of observable behaviors displayed by each mouse over time and quantified these behaviors. there were striking differences between behaviors exhibited by wt and mras−/− males in their roles as residents.fig.  altered social behavior in mras −/− males. a cumulative duration of aggressive behavior by the resident was scored during 10-min resident-intruder tests. mras −/− residents displayed enhanced aggressive behavior towards ko and wtsw intruders but not towards wt or ob intruders. wt/wt vs. ko/ko, p = ; ko/ko vs. ko/wt, p = ; ko/ko vs. ko/ob p = ; ko/wt vs. ko/wtsw, p = ; ko/ob vs. ko/wtsw, p = ; all others not significant. b social investigations by the residents during the first 5 min of the resident-intruder tests were scored. wt/wt vs. ko/ko, p < ; ko/ko vs. ko/ob p = . c social investigations by the intruders during the first five min. of the resident-intruder tests were scored. there were no significant differences for both wt and ko intruders. d digging behavior by the residents was scored during the first 5 min of the resident-intruder tests. p =  for wt/wt vs. ko/ko. e digging behavior by the intruders was scored during the first 5 min of the resident-intruder tests. there were no significant differences for both wt and ko intruders. b–e error bars represent sem. f ‘corner’ behavior was scored for residents, showing more frequent anxiety-related behavior of mras −/− males. p <  for all wt residents vs. all ko residents combined; not significant between individual groups. a–f n =  for all groups except ob where n =  g open field tests: the amount of time mice spent at the center of an open and brightly lit white box was scored over a period of ten min. error bars represent sem, n =  for all groups except n =  for mras −/− males; p = ; females, p = . h mating assays: male mating behavior was scored in 10-min. interactions between combinations of wt and ko males and females. n =  for all groups; p =  for wt/wt vs. ko/ko; p =  for ko/wt vs. ko/ko; not significant for wt/wt vs. wt/ko when paired with intruders of their own genotype, half of the mras−/− resident males exhibited aggression. although the c57bl/6 j mouse strain is considered low to medium aggressive, our wt males did not exhibit aggression during the brief 10 min. assessment period. the difference in aggressive behavior displayed by wt vs. mras−/− residents was significant. we also observed a significant increase in the rate of parental cannibalism of neonates in the mras−/− colony: wt breeder pairs cannibalized of litters, whereas mras−/− breeder pairs cannibalized twice as many litters. cannibalism can be explained by parental aggression, although it can occur due to other factors, such as stress. during resident-intruder tests with isogenic male intruders, mras−/− residents also spent significantly more time investigating intruders compared to wt residents. in contrast, wt residents exhibited significantly increased digging behavior compared to mras−/− residents. thus, in interactions with isogenic males, wt and mras−/− residents displayed social behaviors that differed significantly from one another. curiously, when mras−/− resident males were paired with wt intruders, aggression decreased significantly. mras−/− residents also spent less time investigating wt intruders less, although this did not reach significance. in addition, mras−/− residents did not attack other knockout or transgenic mice on a c57bl/6 j background and displayed significantly decreased social investigations towards these intruders compared to mras−/− intruders. in contrast, wt residents did not change their behavior when paired with mras−/− intruders. thus, the expression of aggression by mras−/− males depended on the genotype of the intruder; mras−/− residents reacted aggressively to mras−/− intruder mice but not to mice that express m-ras, while wt residents either did not make this distinction or did not react to it. mice use pheromonal cues to identify other individuals. thus, we evaluated the behavior of mras−/− residents in the presence of wt intruders that were swabbed with urine from mras−/− males. wtsw intruders did not elicit aggression from wt residents, but elicited aggression from six out of ten mras−/− residents, which was similar to the frequency observed with mras−/− intruders. there difference in aggressive behavior of mras−/− residents towards wt vs. wtsw intruders was significant. aggressive behavior by mras−/− residents may not have been not fully restored with mras−/− social odors because attack duration was shorter on average with wtsw intruders compared to mras−/− intruders. wtsw intruders stimulated social investigations from mras−/− residents at levels that were similar to mras−/− intruders. these results suggested that mras−/− males may produce urinary pheromones that are distinct in quality or quantity from wt males and that could also contain elevated levels of aggression pheromones. however, mras−/− pheromones alone were not sufficient to elicit aggressive behavior because wt mice were not more aggressive towards mras−/− intruders than towards wt intruders. thus, other changes must have occurred in mras−/− males to elicit the aggressive behavioral response. wt residents tended to spend more time investigating mras−/− intruders than wt intruders and exhibited less digging behavior in the presence of an mras−/− intruder, although these differences were not statistically significant. in addition, sniffing and digging behavior displayed by wt intruders was irrespective of the genotype of the resident. thus, wt males followed similar behavioral patterns with isogenic and congenic interaction partners both in their respective roles as residents and as intruders. in contrast, mras−/− males adapted their social behaviors depending on the genotype of the interaction partner when they were residents, but not when they were intruders. inreased anxiety-like behavior in mras−/− mice mras−/− residents frequently retreated to the corner or short edge of the test cage during the resident-intruder test. this ‘corner’ behavior was reminiscent of a fear response, similar to the ‘corner’ behavior observed in open field tests that has been considered anxiety-like behavior. ‘corner’ behavior was very rare with wt residents. compared to wt mice, mras−/− mice also exhibited significantly increased anxiety-like behavior in an open field test, including significantly more time spent in the corners of the open field. the ‘corner’ behavior by mras−/− males in the resident-intruder test was similar in the presence of intruders with different genotypes/odortypes. this suggests that enhanced anxiety-like responses may be a general characteristic of mras−/− males that, unlike sniffing and aggressive behaviors, was not adapted to different opponents. interactions with females we next investigated interactions between male and female mice. we observed a marked increase in male mounting activity among isogenic mras−/− mating pairs relative to isogenic wt mating pairs. ten out of ten mras−/− males engaged in mating behavior within a 10 min. test period, whereas only four of ten wt males did. moreover, all ten mras−/− males spent more than 50 s displaying mating behavior with mras−/− females, whereas only one of ten wt males showed mounting behavior for more than 50 s with wt females. this was likely not due to a general lack of motivation or problems exhibiting mating behavior, as wt and mras−/− males were equally effective at impregnating proestrus females when interactions were allowed to proceed over night. to test the ability of mras−/− males to discriminate between wt and mras−/− females, females of the opposite genotype were paired with the males. intriguingly, while wt males showed about the same amount of mounting behavior with wt and mras−/− females, mras−/− males exhibited significantly reduced mounting activity when paired with a wt female. this result suggests that the display of increased mounting behavior by mras−/− males, like the display of aggression, may depend on the genotype of the interaction partner. in addition, this also suggests that mras−/− females, like mras−/− males, may produce pheromones that are different from the pheromones secreted by wt females. however, these pheromones alone were not sufficient to elicit an increase in mating behavior from wt males, and the increased mounting activity was therefore like aggression a characteristic of mras−/− males. female mating behavior was also assessed. we did not observe lordosis in any of the females tested, which may have been due to the short duration of the assay. we noted that mras−/− females appeared to spent significantly more time investigating mras−/− males compared to wt females investigating wt males. wt females also appeared to spend significantly more time investigating mras−/− males than wt males, while mras−/− females appeared to spend about the same amount of time investigating males of both genotypes. however, female responses were likely affected by male behavior, making interpretation of female behavior difficult. comparable levels of testosterone and sexually dimorphic genes in wt and mras−/− males testosterone is related to aggressive behavior in animals. however, testosterone levels were comparable in the serum and testes of wt and mras−/− males, and those mras−/− residents that displayed territorial aggression did not exhibit significant changes in testosterone levels either. levels of serum estradiol were also similar in wt and mras−/− males. moreover, the expression levels of sexually dimorphic genes that have been implicated in male aggression and male and female sexual behavior were comparable in wt and mras−/− mice. these findings indicated that the lack of m-ras caused changes in social behavior that depended on factors other than sex hormones, sytl irs cckar, or progesterone receptor. they also indicated that mras−/− mice may secrete pheromones that differ from those of wt mice, and the possibility that mras−/− males may have an altered ability to sense pheromones and/or respond appropriately to pheromones.fig.  testosterone levels. a serum testosterone levels in wt and mras −/− males. b total testosterone per mg testis tissue in wt and mras −/− males. c serum testosterone in aggressive and non-aggressive mras −/− males. all data is from six month-old males and shown with sd. a–c there were no significant differences in testosterone levels between wt and mras −/− samples lack of social odor discrimination by mras−/− mice mice spend more time investigating unfamiliar scents over familiar ones and are able to distinguish between very subtle differences in pheromone compositions amongst genetically identical mice. we determined whether wt and mras−/− mice would discriminate between wt and mras−/− male urinary pheromones in the absence of the owner of the scent. as expected, wt females spent significantly greater time and frequency investigating the unfamiliar mras−/− male urine spots relative to wt urine spots. similarly, wt males tended to spend more time investigating mras−/− urine spots, although this did not reach statistical significance. in contrast, both mras−/− males and females spent about the same amount of time investigating wt and mras−/− male urine spots, with mras−/− males exhibiting a slight but significant preference for mras−/− urine spots. we also tested the ability of wt and mras−/− fathers to recognize their own adult offspring. while wt fathers, as predicted, spent significantly more time investigating the alien isogenic offspring than their own offspring, mras−/− fathers spent the same amount of time investigating both their own and the alien isogenic offspring.fig.  evidence for defective vomeronasal function in mras −/− mice. a urine spot investigations. wt mice, but not mras −/− mice, spent more time investigating an unfamiliar over a familiar urine spot and visited the unfamiliar spot more frequently. data is shown with sem; n =  for males, n =  for females; one-sample t-tests: wt females: p = , p = ; mras −/− males: p = , p = ; all others, not significant. b parental offspring recognition test. mras −/− fathers, in contrast to wt fathers, were unable to discriminate between their own offspring and an unrelated isogenic mouse. one-sample t-tests: wt: p = , n = 7; mras −/−: not significant, n = 8 these results alluded to two possible underlying problems in mras−/− mice. that they were unable to recognize familiar pheromones or to discriminate between pheromones from isogenic and congenic mice suggests that pheromone detection, possibly by the vomeronasal organ, may be compromised in mras−/− mice. however, mras−/− males clearly displayed different behaviors with wt and mras−/− interaction partners, which paradoxically indicated that pheromones were detected by mras−/− mice. basic olfaction by the main olfactory bulb, as determined by a buried food test, and gross olfactory bulb anatomy were similar in wt and mras−/− males. although immediate detection of pheromones may be intact, pheromone processing and subsequent olfactory learning could be defective in mras−/− mice. this may have rendered them unable to generate appropriate interpretations and social behaviors in response to pheromones. alternatively, mras−/− males may produce distinct urinary pheromones that evoke increased interest in mras−/− mice. we addressed both possibilities in subsequent experiments. known mechanisms that contribute to olfactory learning are similar in wt and mras−/− mice we explored several processes that are known to affect olfactory learning to determine if they may underlie an apparent defect in vno function in mras−/− mice. first, we tested acute pheromone sensing and signaling by g protein-coupled receptors in vno neurons. exposure to alien pheromones leads to the induction of the immediate-early genes, c-fos and egr-, and gαo function in the vno is required for aggressive behavior ). we exposed wt and mras−/− males to soiled bedding from either isogenic or congenic mice for 30 min. and analyzed ieg induction in vno tissue. exposure to either bedding source led to a significant upregulation of c-fos. egr- expression was also induced although not significantly. there were no significant differences in the levels of induction of c-fos and egr- between wt and mras−/− mice, which suggested that there were also no differences in g-protein coupled signaling by vomeronasal receptors leading to induction of these two key genes. thus, acute pheromone sensing was likely intact in mras−/− mice, which supports our conclusion from the results shown above.fig.  ieg induction, expansion of neural progenitors, and regulation of th. a, b c-fos and egr- gene induction in the vno after exposure to alien male bedding for 30 min. control mice remained in their own cage, ‘iso’ and ‘con’ refers to exposure to soiled bedding from isogenic or congenic mice, respectively. data is shown with sd, n =  for all groups except mras −/− exposed to ‘iso’, n =  and mras −/− exposed to ‘con’, n = 4; no significant effect of genotype for either ieg. c, d the average number of ns generated and average ns sizes at varying concentrations of egf were similar for wt and mras −/− neural progenitors. there were no significant differences between wt and mras −/− samples; n =  for experiments in c, n =  for experiments in d, error bars represent sd. e stimulation of prlr- or prlr- with prolactin resulted in weak activation of m-ras but with different kinetics; n =  for prlr- samples; n =  for prlr- samples; error bars represent sem. f prolactin did not affect the number of ns generated. g prolactin slightly stimulated the proliferation of wt or mras −/− neural progenitors to similar levels; n =  wt ctrl, n =  wt + prl, n =  mras −/− ctrl, n =  mras −/− + prl; data shown with sem. h tyrosine hydroxylase gene expression in the olfactory bulbs of mated and plugged females. data is shown with sd, n =  for all groups except mras −/− dpc where n = 3; no significant effect of genotype epidermal growth factor is the major growth factor driving the expansion of neural progenitor cells in the svz. as m-ras is potently activated by egf and is highly expressed in the brain, including the svz, the rostral-migratory stream, and the olfactory bulbs, we evaluated egf responsiveness of svz neural progenitors from wt and mras−/− mice in a neurosphere assay. however, the number of ns generated and their expansion with egf, including long-term proliferation, was virtually identical in wt and mras−/− cells, which suggests that expansion of neural progenitors during olfactory learning is likely similar in wt and mras−/− mice. prolactin acts through prlr isoforms of the cytokine receptor family and so was predicted to stimulate m-ras activation. indeed, transcript variants prlr- and prlr- conferred weak but consistent m-ras activation with different kinetics. prlr- which has the shortest intracellular domain of the prlr isoforms, did not activate m-ras. the addition of prolactin to ns assays did not affect the number of ns generated from wt or mras−/− neural progenitors, but the average size of both wt and mras−/− ns increased slightly, and to a similar extent, with addition of prolactin to the cultures, which indicated that prolactin mildly stimulated the proliferation of nsc. interestingly, while svz tissue expressed detectable levels of all three prlr isoforms, with the longest isoform prlr- being the most abundant, we were unable to detect any prlr transcripts in cultured nsc. thus, the proliferative stimulation likely occurred early in vitro, either before the neural progenitors downregulated prlrs, or after supporting cells died off. nevertheless, prolactin stimulated the expansion of neural progenitor cells to similar extents in both wt and mras−/− cells. another factor that can contribute to changes in social behavior is a change in motivation. interest in mating and display of aggression can be considered motivation-based behaviors that are associated with reward, and both are associated with dopamine production or signaling. tyrosine hydroxylase is the key enzyme in dopamine biosynthesis; it can be used as an indirect measure of dopamine production. we analyzed the changes in expression levels of th upon mating in the olfactory bulbs of female mice. th rna levels changed significantly, dropping early after mating and subsequently increasing to steady-state levels or slightly above. the changes in th expression levels were similar in wt and mras−/− mice, which suggests that dopamine metabolism was likely not significantly different between these mice. the urinary metabolite profile of mras−/− males may be distinct from wt males compounds that function as pheromones can be volatile or non-volatile chemicals, or can be proteins. to determine whether wt and mras−/− male urine differ in protein content, we tested randomly collected urine samples by sds electrophoresis and coomassie stain. the concentration of 21 kda major urinary proteins, which contain and may bind to pheromones, was slightly higher in mras−/− males. however, the levels of darcin, which was identified as an attractive pheromone, were similar in wt and mras−/− males. next, we analyzed urine samples by nmr and mass spectrometry for urinary metabolites. several metabolites were dysregulated in mras−/− urine. for example, glycerate was increased -fold and succinate was decreased -fold. in addition, some components of lipid metabolism were detected at different levels in wt and mras−/− urine. sixty-four percent of the phosphatidylcholines and 43 % of the lysophosphatidylcholines tested were increased at least two-fold, and 44 % of sphingomyelins were downregulated greater than two-fold in mras−/− urine compared to wt urine. interestingly, mras−/− male urine contained twice as much trimethylamine as wt male urine. trimethylamine has been identified as an attractive chemosignal in mouse urine. the metabolomics analysis was performed only once with urine samples pooled from three males each and did not include many volatile compounds. however, the results from the resident-intruder tests may support the conclusion that the urinary metabolite/pheromone profiles likely differ between wt and mras−/− males, because mras−/− residents reacted differently to wt vs. wtsw or mras−/− intruders.fig.  major urinary proteins and darcin. urine was randomly collected from six wt and mras −/− male mice each. two µl of urine was loaded onto a 15 % sds gel per sample. gels were stained with coomassie blue to visualize protein. mups are observed at kd, darcin at approximately 16- kd table  urine metabolomics analysis wt and mras −/− male urine samples were analyzed by nmr and direct-injection mass spectrometry. total number of metabolites analyzed by nmr: 65; di-ms: some differences in relative metabolite quantities were confirmed by gas chromatography -ms analysis: glyceric acid: +; l-tyrosine: +; succinic acid: −; hippuric acid: − in summary, while none of the known factors contributing to pheromone sensing and olfactory learning appears to be compromised in mras−/− mice, it is possible that other, unexplored mechanisms may be affected by the absence of m-ras. moreover, it is likely that distinct pheromones produced by mras−/− males additionally affect their social behavior. discussion we uncovered changes in social behavior in mice lacking m-ras, and used a graphical representation of dynamic social interactions, a behavior heatmap, to visualize differences in the ways wt and mras−/− mice dynamically interact with each other. mras−/− males exhibited higher levels of territorial aggression with other mras−/− males and spent more time than wt males to investigate an intruder; often, these prolonged investigations culminated in a physical attack. mras−/− mice displayed less digging behavior but increased fear-related responses such as the ‘corner’ response. in mating assays with females, mras−/− males more readily engaged in mating behavior compared to their wt counterparts. thus, mras−/− males showed social behaviors that were strikingly different from wt males. the underlying causes for aggressive behavior have been the subject of intense investigations, particularly in humans. although genetics and differentially regulated brain chemistry or neuronal circuits may play a role, aggression is also strongly influenced by experience and increases with positive reinforcement. another determinant of aggressive behavior is the interaction partner with his unique stimulus properties/characteristics. some mouse strains elicit higher levels of aggression than others, and hormone status and gender of the opponent can also affect aggressive responses. it is likely that olfactory cues emanating from the opponents induce aggressive behaviors in resident males. many different knockout mouse strains are known to exhibit changes in aggression levels. to our knowledge, our study is the first to show that an increase in aggressive behavior depended on two factors: the absence of mras in the resident and the absence of a single gene in the intruder, mras, unless compensatory changes in expression levels of other genes occurred during development. our data supports that differentially secreted pheromones by mras−/− mice may contribute to the occurrence of aggression. if wt and mras−/− mice indeed emit different social odors, a wt intruder would be less familiar than an mras−/− intruder for an mras−/− resident male who had grown up in the presence of mras−/− but not wt social odors. thus, the prediction was that the wt intruder should have suffered greater aggression. that the opposite occurred was surprising and will be further discussed below. the increases in aggression and mounting activity in mras−/− males were unrelated to levels of testosterone or estradiol and expression levels of genes that are considered sexually dimorphic. the serotonin system also contributes to the regulation of social interactions, including aggression, in humans and mice. mice deficient in 5-ht1b exhibit increases in aggressive behavior, and greater serotonin activity positively affects social interactions in humans and animals. an optimal level of serotonin signaling is required during brain development for normal behavior in adulthood, and anxiety is associated with reduced function. it will be of interest to determine if mras−/− mice exhibit changes in the serotonin system that could promote an increase in aggression and/or in fear-related behavior. enhanced aggression and enhanced anxiety could contribute to an inability to appropriately assess contextual risk and adjust behavior accordingly. wt and mras−/− resident males also differed in their digging behavior. digging was a characteristic of wt but not mras−/− residents. digging could represent an attempt to escape, perhaps in search of shelter. we interpreted digging during the resident-intruder test as an expression of frustration. thus, it is possible that wt and mras−/− males may express frustration differently. another interesting observation emerged from our studies: mras−/− mice failed to discriminate both between highly similar, isogenic urinary pheromones, and even between pheromones from congenic and isogenic mice, where greater differences in pheromone composition would be expected. this could be interpreted as evidence for a defect in acute pheromone processing in mras−/− mice. the problems could be within the vno and affect immediate detection. however, c-fos induction in the vno in response to social odors was similar in wt and mras−/− males. alternatively, processing of pheromone information downstream of the vno may be defective. in particular, the medial amygdala integrates social odor cues from signals from both the main and accessory olfactory systems. although mras−/− mice did not discriminate between pheromones in the absence of the owners of these scents, they did discriminate between isogenic and congenic mice when the animal was present and available for social interactions, and exhibited enhanced aggression and mating behavior only towards isogenic partners. this seemed paradoxical, but suggested that pheromones were detected and that acute olfactory and/or vomeronasal function was intact in mras−/− males. there are several possible explanations. first, independent of pheromones or their mechanisms of detection and processing, mice may be able to detect and interpret non-olfactory contextual information. this would depend both on the perception of the resident and on the behavior of the intruder. the intruder with his own characteristics will affect resident behavior. for example, wt residents may detect differences in motor patterns or anxiety in mras−/− intruders and may not feel the need to attack, and mras−/− residents paired with mras−/− intruders could sense each other’s anxiety to produce changes in behavioral patterns. however, this is likely only a minor factor because mras−/− residents exhibited similar levels of aggression and social investigations with mras−/− intruders and wt intruders when the latter were swabbed with mras−/− urine, but they did not attack unswabbed wt intruders even though the wt intruders did not change observable behaviors whether they were swabbed with urine or not. second, we cannot rule out that the olfactory and/or social recognition processes may be malfunctioning in mras−/− mice, as we have only addressed a subset of molecular correlates. immediate detection of pheromones was intact in mras−/− males, as assessed by the induction of c-fos upon pheromone exposure and the differential behavioral responses with wt and mras−/− territorial intruders. in addition, neural progenitor cells of mras−/− mice had the same capacity to respond to an egf or prolactin stimulus cells from wt mice. there are conceivably many more cellular and biochemical players that contribute to a highly complex learning process. for example, we have not addressed the possibility that mras−/− mice may exhibit epigenetic modifications, which could indicate that they inherited an olfactory experience from their ancestors. moreover, hormones such as oxytocin, vasopressin, and serotonin have been implicated in the molecular basis of social recognition. in particular, the closely related oxytocin and vasopressin may be involved in longer-term social recognition and modulation of behavioral responses in rodents. mice lacking oxytocin do not display an increase in anxiety and a deficiency in the vasopressin receptor, v1ar, may result in male-specific reduction in anxiety levels. in contrast, both male and female mras−/− mice showed higher levels of anxiety. vasopressin may play dual roles in the regulation of aggressive behavior, facilitating or inhibiting aggression depending on the specific site of release in the brain. it will be worthwhile to examine the contributions of these hormones to the behavioral changes in mras−/− mice. in addition, changes in other hormones and their receptors could drive the changes in social recognition and the associated differences in behavior. third, despite the evidence that points to a possible defect in vno function, we cannot exclude a role for pheromones, or the processing of pheromones, in mras−/− mouse behavior. we observed that mras−/− mice may differentially produce several urinary metabolites compared to wt mice. we are currently exploring whether this may correlate with an enlargement of urinary bladders that frequently occurs in mras−/− males and seems to worsen with age. one metabolite that was elevated in mras−/− urine, trimethylamine, is a known pheromone. by virtue of its attractive properties, trimethylamine may facilitate social interactions between mice. interestingly, high concentrations of trimethylamine can induce an aversive response in mice. the receptor for trimethylamine is taar; it will be of interest to determine if taar levels are dysregulated in mras−/− mice. the altered secretion of trimethylamine and other urinary metabolites suggest that is very likely that the pheromone profile of mras−/− mice differs from wt mice. it was shown that mice that are deficient in oxytocin or estrogen receptor β produce urinary substances that facilitate an aversive response. it is possible that mras−/− mice could produce distinct pheromones, such as trimethylamine, that may be perceived as enticing to both wt and mras−/− mice and affect behavioral responses. darcin is another urinary protein that is perceived as attractive, although its levels were similar in wt and mras−/− male urine. the attraction of an mras−/− male to an as yet unidentified, mras−/−-specific pheromone could cancel out the urge to explore a novel urine spot produced by a wt male when mras−/− urine is present at the same time, and could further stimulate a wt male to explore an mras−/− urine spot. likewise, the unknown pheromone could restore exploration and attack of wt intruders by mras−/− residents when the former were swabbed with mras−/− urine, and could explain why mras−/− fathers investigate both their own and an alien offspring equally. the results from the offspring recognition tests, where test subjects were only exposed to airborne pheromones, suggest that the unknown, attractive pheromone could be volatile. thus, the apparent defect in vno function may be an artifact produced by an enticing pheromone secreted by mras−/− mice. however, even though urinary metabolites and pheromones may be differentially secreted by mras−/− mice, these alone were not sufficient to evoke an aggressive response or increased mating behavior by wt males, and additional mechanisms that could be involved with the processing of olfactory information must be dysregulated in mras−/− males for aggression and interest in mating to surface more readily. so far we have only explored the acute detection of pheromones in the vno and the expansion of neural progenitors and found that these were similar in wt and mras−/− mice. however, other molecular pathways downstream of the induction of iegs, and/or as yet unknown molecular mechanisms and neural circuits that contribute to the processing of olfactory information could be affected by the absence of m-ras. fourth, in combination with genetics and pheromones, learning will also affect behavior. without a learning process, memory cannot be established. interestingly, behavioral performance of neonates in an associative olfactory learning task was shown to depend on transcript levels of rasgrf which is an activator of m-ras. in addition, the mras gene is upregulated upon spatial discrimination learning in rats. we observed that mras−/− mice show a slight delay in spatial learning in the morris water maze test, which was likely not due to an impairment in motor function. however, they caught up after eight training sessions and the consolidation of reference memory was similar to wt mice. if mras needs to be upregulated for an optimal hippocampus-dependent spatial learning task, it may also need to be increased for optimal olfaction- or pheromone-dependent social recognition, and the behavioral changes we have observed in mras−/− mice could be indicative of a slight learning deficit in general. thus, mras−/− males could take longer to process pheromone information emitted by unfamiliar wt intruders and we cannot rule out that mras−/− resident males, when given more time, might attack wt intruders eventually. likewise, mras−/− fathers could take longer to process the similar pheromones produced by their own and alien isogenic offspring, and may require more time to determine that there are differences. alternatively, it is also possible that there may be defects in the formation of social odor memory. the oxytocin and vasopressin systems have roles in social odor memory consolidation, and more experiments are required to address the possibility of their involvement and to distinguish between short-term recognition and long-term memory formation. in addition, mras−/− pups could be fostered to wt parents in order to test whether there are problems with olfactory recognition or if there might be a component of faulty experiential learning through a parenting defect. finally, it is possible that the observed behavioral changes in mras−/− males could have been a consequence of ontogenetic processes caused by the deletion of the mras gene, resulting in changes in expression levels of other genes. in addition, given the similarity to other proteins of the ras family of gtpases, especially r-ras, tc and the p ras proteins, it is conceivable that these proteins could have taken over some of the normal functions of m-ras, but could have skewed signaling pathways through slightly different effector usage. roles for ras proteins in social behavior have not yet been reported, although neurofibromatosis, which inactivates ras proteins by stimulating their gtpase activities, has been implicated in social recognition in mice. it will be of interest to determine whether mice with deletions of other ras proteins exhibit behavioral changes; this could provide insight into possible compensatory mechanisms in mras−/− mice.  in summary, mras−/− males exhibited changes in social behavior that included increased territorial aggression, increased mating behavior, and fear-like responses. these changes were likely caused by a combination of several factors. pheromones may be differentially produced and/or differentially sensed and interpreted by mras−/− mice. these pheromones could interfere with the correct identification of other individuals, which in turn could affect olfactory learning and result in an inability to respond appropriately to social stimuli. however, the behavior changes in mras−/− males cannot be explained by differences in pheromones alone, as these were not sufficient to elicit aggression or enhanced mating behavior from wt males. other mechanisms, perhaps a signal transduction pathway that is under- or overactive by the absence of m-ras must be altered to predispose mras−/− mice to display altered behavior. as social interactions in higher organisms are highly complex this will require more detailed investigations into the molecular pathways and neural circuits that m-ras may affect. 
although this could indicate that vomeronasal function and/or olfactory learning may be compromised in mras−/− mice, these observations were not fully consistent with the differential behavioral responses to wt and mras−/− interaction partners by mras−/− males. in their interactions with other males, mras−/− males exhibited high levels of territorial aggression and social investigations, and increased fear-related behavior. curiously, increased aggression and mating behaviors were only observed when mras−/− males were paired with mras−/− partners, but were significantly reduced when paired with wild-type mice. olfactory and/or social learning processes may thus be compromised in mras−/− mice. in addition, induction of c-fos upon pheromone exposure or in response to mating was similar in wt and mras−/− mice, as was the ex vivo expansion of neural progenitors with egf. however, urinary metabolite profiles differed between mras−/− and wt males. since mice use pheromonal cues to identify other individuals, we explored the possibility that pheromone detection may be altered in mras−/− mice. the molecular mechanisms that determine social behavior are poorly understood. here, we report that the absence of the small gtpase m-ras affects social behavior in mice. the changes in behaviors displayed by mras−/− mice are likely due to a complex combination of factors that may include an inherent predisposition to increased aggression and sexual behavior, and the production of distinct pheromones that could override the preference for unfamiliar social odors. unlike wt mice, mras−/− did not show a preference for exploring unfamiliar urinary pheromones or unfamiliar isogenic mice. pheromones play a critical role in social recognition in most animals, including mice, but how these are converted into behavioral responses is largely unknown. this indicated that acute pheromone detection and processing was likely intact. r-ras3social recognitionvnoanxietybehavior heatmapissue-copyright-statement© the author 2015
 the molecular mechanisms that determine social behavior are poorly understood. pheromones play a critical role in social recognition in most animals, including mice, but how these are converted into behavioral responses is largely unknown. here, we report that the absence of the small gtpase m-ras affects social behavior in mice.  in their interactions with other males, mras−/− males exhibited high levels of territorial aggression and social investigations, and increased fear-related behavior. they also showed increased mating behavior with females. curiously, increased aggression and mating behaviors were only observed when mras−/− males were paired with mras−/− partners, but were significantly reduced when paired with wild-type mice. since mice use pheromonal cues to identify other individuals, we explored the possibility that pheromone detection may be altered in mras−/− mice. unlike wt mice, mras−/− did not show a preference for exploring unfamiliar urinary pheromones or unfamiliar isogenic mice. although this could indicate that vomeronasal function and/or olfactory learning may be compromised in mras−/− mice, these observations were not fully consistent with the differential behavioral responses to wt and mras−/− interaction partners by mras−/− males. in addition, induction of c-fos upon pheromone exposure or in response to mating was similar in wt and mras−/− mice, as was the ex vivo expansion of neural progenitors with egf. this indicated that acute pheromone detection and processing was likely intact. however, urinary metabolite profiles differed between mras−/− and wt males.  the changes in behaviors displayed by mras−/− mice are likely due to a complex combination of factors that may include an inherent predisposition to increased aggression and sexual behavior, and the production of distinct pheromones that could override the preference for unfamiliar social odors. olfactory and/or social learning processes may thus be compromised in mras−/− mice. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords r-ras3social recognitionvnoanxietybehavior heatmapissue-copyright-statement© the author 2015 