Charlotte M. Nevison

The ownership signature in mouse scent marks is involatile.

Male house mice advertise their territory ownership through urinary scent marks and use individual–specific patterns of major urinary proteins (MUPs) to discriminate between their own scent and that of other males. It is not clear whether recognition occurs through discrimination of the non–volatile proteins or protein–ligand complexes (direct model), or by the detection of volatile ligands that are released from MUPs (indirect model). To examine the mechanism underlying individual scent mark signatures, we compared investigatory and countermarking responses of male laboratory mice presented with male scent marks from a strain with a different MUP pattern, when they could contact the scent or when contact was prevented by a porous nitrocellulose sheet to which proteins bind. Mice investigated scent marks from other males whether these were covered or not, and biochemical analysis confirmed that the porous cover did not prevent the release of volatiles from scent marks. Having gained information through investigation, mice increased their own scent marking only if they had direct contact with another males urine, failing to do this when contact was prevented. Individual signatures in scent marks thus appear to be carried by non–volatile proteins or by non–volatile protein–ligand complexes, rather than by volatiles emanating from the scent.

MHC odours are not required or sufficient for recognition of individual scent owners.

To provide information about specific depositors, scent marks need to encode a stable signal of individual ownership. The highly polymorphic major histocompatibility complex (MHC) influences scents and contributes to the recognition of close kin and avoidance of inbreeding when MHC haplotypes are shared. MHC diversity between individuals has also been proposed as a primary source of scents used in individual recognition. We tested this in the context of scent owner recognition among male mice, which scent mark their territories and countermark scents from other males. We examined responses towards urine scent according to the scent owners genetic difference to the territory owner (MHC, genetic background, both and neither) or genetic match to a familiar neighbour. While urine of a different genetic background from the subject always stimulated greater scent marking than own, regardless of familiarity, MHC-associated odours were neither necessary nor sufficient for scent owner recognition and failed to stimulate countermarking. Urine of a different MHC type to the subject stimulated increased investigation only when this matched both the MHC and genetic background of a familiar neighbour. We propose an associative model of scent owner recognition in which volatile scent profiles, contributed by both fixed genetic and varying non-genetic factors, are learnt in association with a stable involatile ownership signal provided by other highly polymorphic urine components.

Understanding behaviour: the relevance of ethological approaches in laboratory animal science

Applied ethology has traditionally focused on farm animal species, whereas there has been much less research directed at understanding the behaviour of laboratory animals in relation to their use as models in research. In this paper, we identify four areas in which ethological approaches could help improve the welfare of laboratory rodents while at the same time enhancing the validity of research based on them. These areas are: (1) the effects of selective breeding and gene manipulations on the animals’ ability to cope with the laboratory environment; (2) the effects of barren housing conditions on behaviour and the mechanisms underlying normal control of behaviour; (3) the sensory perception of the laboratory environment by the animals; and (4) the applicability of standard behavioural tests and the potential for improving them by taking animals’ species-specific characteristics into account. Given the current increase in the use of rodents in the life sciences, these four areas represent promising areas of future research in applied animal behaviour science. # 2003 Elsevier Science B.V. All rights reserved.

Why do male ICR(CD-1) mice perform bar-related (stereotypic) behaviour?

Prolonged interaction with cage bars by captive mammals (usually classed as stereotypic) may reflect poor welfare. Such behaviour may arise from motivation to investigate the external environment or to escape captivity. However, these hypotheses have not been explicitly tested. We raised mice, Mus musculus, to adulthood in modified laboratory cages with two sets of bars at the top and side of the cage. One set provided a potential escape route, and half of each set was backed by Perspex to reduce cues from the external environment. We predicted where mice should interact with the bars according to their motivational priorities. Body weights were recorded weekly to study the relationship between physical development and bar-related behaviour. Serum corticosterone was measured to monitor the effect of bar-related behaviour on stress physiology. Mice preferred to interact with bars where external cues were detectable. As adults, mice responded more to the bars providing a potential exit, though this was affected by the exit location. Corticosterone titres were higher in mice whose potential exit was situated at the cage top. Response to the bars was apparently restricted by the physical development of mice, particularly among those whose potential exit was situated in the cage top.

The consequence of inbreeding for modulating social relationships between competitors

Abstract Extreme inbreeding is likely severely to compromise an animal’s ability to discriminate between individuals that are genetically very similar or identical, and thus to assess their familiarity and kinship with conspecifics. We tested whether male laboratory mice of the outbred ICR strain and the inbred BALB/c strain could discriminate between familiar companions and unfamiliar males of their own strain, or between unfamiliar mice from their own or the other strain, by examining both responses to the males’ scents and competitive aggression during direct interactions. Outbred ICR males spent more time investigating odours of unfamiliar males and were more aggressive towards unfamiliar males than towards familiar companions of their own strain. BALB/c males failed to discriminate between familiar and unfamiliar males of their own strain, showing little aggression to both. In contrast, most BALB/c males (17/24) were aggressive towards unfamiliar ICR males and investigated their scent for longer than that from their own strain. The low level of aggression towards unfamiliar own-strain males suggests that the inbred males failed to recognise that unfamiliar individuals were not their familiar cagemates. They were unable to discriminate between different individuals from their own strain using either chemical or other additional cues of individual identity available during direct interaction.

Information in Scent Signals of Competitive Social Status: The Interface Between Behaviour and Chemistry

From an evolutionary viewpoint, signals generally should be reliable or honest (Zahavi, 1987; Johnstone, 1997). Animals can gain a number of advantages from advertising high competitive ability to potential mates and to other competitors, particularly males which often compete strongly for mating opportunities (Andersson, 1994). Animals often prefer high quality mates that will increase the fitness of their offspring, both because of genetic benefits (through good genes or Fisherian selection) and because parents of high competitive ability often provide better resources and protection. Competitors will also gain an advantage if potential challengers withdraw from, or otherwise avoid, aggressive encounters with an opponent of high fighting ability. There is thus strong selection pressure on signallers to advertise high social status and competitive ability to others. However, receivers will only gain an advantage from responding to such signals if these are reliable indicators of the signaller’s competitive ability. Females that mate with low quality males that dishonestly signal high competitive ability will gain no advantage for their offspring, while males that withdraw from agonistic encounters with poorer competitors will be disadvantaged. There is thus strong selection on receivers to respond only to honest signals that are resistant to cheating, and therefore for high quality animals to provide such reliable information in signals as these will be effective in attracting mates and deterring competitors.

Mice, Mups and Myths: Structure-Function Relationships of the Major Urinary Proteins

The presence of high concentrations of protein in the urine of many mammals is often a pathological event reflecting a failure of the renal mechanisms to prevent passage of plasma proteins through the glomerular filter, or for recovery of protein in the proximal tubule. In adult humans, for example, the normal urinary protein concentration is of the order of 50μg/ml, a value that serves to emphasise the remarkable protein output in the urine of mice, and to a lesser extent rats. The average protein concentration in the house mouse (Mus domesticus) reaches between 200 and 2000 times that of a normal human, at concentrations of the order of 30mg/ml. Over 99% of this protein comprises the Major Urinary Proteins, a group of 18–20kDa proteins, synthesised in the liver, secreted into plasma and subsequently passed through the glomerular filter into the urine as it is elaborated.

The “scents” of ownership

Scent marks need to encode a reliable signal of ownership to inform about the specific owner. Although tests of discrimination have told us much about the abilities of animals to detect differences in conspecific scents deriving from a wide range of sources, we know little about the components involved in scent ownership recognition or individual recognition when animals meet because this requires functional tests of individual recognition. Ownership signals in scent marks need to be stable and persistent, ideally genetically determined and sufficiently polymorphic. Recent work from our laboratory, using functional tests of scent mark recognition, suggest that the pattern of polymorphic MUPs in the urinary scent marks of male house mice provides an ownership signal. The ownership signal is involatile, requiring investigatory contact with the scent source, and involves either involatile complexes between MUPs and their bound odorants or the MUPs themselves, probably detected through the vomeronasal system. However, mice also detect non-MUP related differences in urinary volatiles. We propose a model of learnt association between involatile and volatile components that would allow mice to recognize previously encountered volatile profiles from familiar individuals or animals of the same sex without requiring close contact investigation. Investigation of fresh scent marks deposited around the environment would allow animals to update the proposed association between an individual’s stable involatile profile with any changes in its volatile profile. Further research is required to test this model and to establish its generality in other mammalian species.

The Role of Urinary Proteins and Volatiles in Competitive Scent Marking Among Male House Mice

Male house mice (Mus domesticus), like many other male mammals, advertise their competitive dominance and ability to defend territories by depositing numerous urinary scent marks throughout their territory (reviewed by Rails, 1971; Johnson, 1973; Gosling, 1982, 1990; Hurst, 1987). Male mice also increase their rate of marking near any competing scent marks from other males, a behaviour termed counter-marking (Hurst, 1990, 1993; Hurst and Rich, 1999). Because only those males successfully dominating their territory can ensure that their own marks are always the freshest and predominant in the area, other males can use the temporal and spatial deposition dynamics of male scent marks to assess territory ownership and competitive ability (see Hurst et al., this volume). Perhaps more importantly, female mice can also use these scent marks to assess the quality of potential mates, preferring dominant male territory owners that counter-mark scent mark challenges from competitors and which ensure that their own marks are always the freshest (Rich and Hurst, 1999).

Effects of Inbreeding and Social Status on Individual Recognition in Mice

Individual recognition modulates social behaviour between conspecifics, enabling an animal to assess its familiarity and kinship with other individuals (Barnard et al., 1991; Barnard and Aldhous, 1991). Wild mice identify each other through individually unique urinary odour cues that are determined, at least in part, by genetic differences (Eggert et al., 1996). By depositing these unique odour cues as scent marks, mice provide signals of their presence and social status (see Hurst et. al., this volume). Dominant males deposit scent marks at high frequency as a sign of their competitive quality and current territorial ownership, and increase their rate of scent marking where they encounter competing scent marks from other males in their territory. Countermarking of scent marks from other males by dominant males thus provides a specific test that the mice have recognised scent marks as being derived from another individual.