The influence of photoperiod and sex on lipopolysaccharide-induced hypoactivity and behavioral tolerance development in meadow voles (Microtus pennsylvanicus)

Abstract

Lipopolysaccharide (LPS), the minimal immunogenic component of Gram-negative bacteria, is released during infection and causes a variety of sickness behaviors including decreased locomotor activity. This study considered how photoperiod and sex influence the effects of LPS in the meadow vole, Microtus pennsylvanicus. Male and female voles were housed under either reproductively stimulatory (long day: 16 h) or inhibitory (short day: 8 h) photoperiods. On Days 1 and 8, voles were injected with LPS (200 μg/kg, i.p.) or saline vehicle and locomotor activity was assessed 2 h later in an automated open field for 1 h. The first exposure to LPS caused significant decrements in locomotor activity in all LPS-treated groups, regardless of photoperiod or sex. On Day 8, both short day males and females exhibited behavioral tolerance to LPS, no longer displaying significant activity decrements. In contrast, long day females reinjected with LPS on Day 8 still exhibited significant hypoactivity on all locomotor measures. Similarly, long day males also appeared to exhibit a sustained expression of sickness behaviors on Day 8. In long day females, higher circulating progesterone levels were associated with an attenuated rate of tolerance formation to LPS. The present findings support the winter immunoenhancement hypothesis, which states that small mammals which undergo severe seasonal fluctuations undergo compromised immune functioning during the breeding season, and further indicate a potential role for progesterone in modulating these seasonal immune fluctuations in females.

Introduction

Lipopolysaccharide (LPS), derived from the cell wall of Gram-negative bacteria, is commonly used to model infection in laboratory animals and, more recently, in humans. Systemic administration of LPS causes a host of stereotypical behavioral responses to occur which include, but are not limited to, reduced activity (Kozak et al., 1994, Linthorst et al., 1995, Linthorst et al., 1996, Linthorst et al., 1997, Yirmiya, 1996, Avitsur et al., 1997, Huang et al., 1999, Engeland et al., 2001b), reduced intake of both food and water (O’Reilly et al., 1988, Langhans et al., 1991, Langhans, 1996, Swiergiel et al., 1997, Huang et al., 1999, Cross-Mellor et al., 2000) and increased sleep (Meltzer et al., 1989). These “sickness behaviors” are centrally mediated following the peripheral release of proinflammatory cytokines (interleukin [IL]-1β and tumor necrosis factor [TNF]-α). Physiological and behavioral tolerance to repeated injections of LPS typically forms rapidly, often after a single exposure (O’Reilly et al., 1988, Langhans et al., 1991, Roth et al., 1997, Porter et al., 1998, Tripp et al., 1998), and is a result of the decreased synthesis and release of (Mathison et al., 1990, Knopf et al., 1994, Zeisberger and Roth, 1998), and a decreased responsiveness to (He et al., 1992), these pro-inflammatory cytokines.

It is now well accepted that such sickness behaviors are adaptive and help an organism to cope with bacterial infection. For instance, anorexia and hypodipsia reduce metabolic expenditures associated with digestion, decrease circulating blood plasma levels of iron and zinc (which bacteria require for growth and proliferation) and reduce the need for an animal to be active. Moreover, reducing activity levels when ill conserves energy, aids in the maintenance of fever and reduces the chance of predation in the wild (Hart, 1988). As reduced locomotion is both a common sickness behavior and an adaptive response to infection, locomotor activity provides a useful index for the assessment of both the effects of, and responses to, acute bacterial infection in animals.

It has been shown repeatedly that intraperitoneal (i.p.) LPS administration reduces locomotor activity levels in laboratory male (Linthorst et al., 1995, Linthorst et al., 1996, Linthorst et al., 1997, Yirmiya, 1996, Avitsur et al., 1997, Huang et al., 1999) and female (Avitsur et al., 1997, Engeland et al., 2001a, Engeland et al., in press) rats and in male mice (Kozak et al., 1994, Engeland et al., 2001b). LPS has also been shown to have a number of physiological and behavioral effects in other species of rodents, including microtine rodents such as the meadow vole (Microtus pennsylvanicus). These effects include increased splenocyte proliferation (Klein and Nelson, 1998), activation of the hypothalamic–pituitary–adrenal (HPA) axis, IL-1β release and reduced affiliative behaviors (Klein and Nelson, 1999). However, to date, activity modifications as a result of bacterial infection and sickness have not been systematically examined in meadow voles of either sex.

A sexual dimorphism, chiefly mediated by gonadal hormones, is apparent in immune functioning (Gaillard and Spinedi, 1998, Lahita, 2000). Specifically, it has been demonstrated in rats and mice that testosterone has immunosuppressive effects on both the humoral and cell-mediated arms of immunity and inhibits macrophage activation, whereas estrogen has immunoenhancing effects on humoral immunity and immunosuppressive effects on cell-mediated immunity (McCruden and Stimson, 1991, Giglio et al., 1994, Wichmann et al., 1997, Savita and Rai, 1998). However, compared to males, females have overall enhanced functioning of both arms of immunity (Schuurs and Verheul, 1990, Zuk and McKean, 1996). Despite these findings, relatively little is known about sex differences in the expression of sickness behaviors.

There is evidence from several species of voles and various other species of rodents to suggest that immune functions are modulated by both reproductive status (Nelson et al., 1999, Lochmiller and Moshkin, 1999, Nelson and Klein, 2000, Sinclair and Lochmiller, 2000, Bilbo et al., 2002) and gonadal hormone levels (Schuurs and Verheul, 1990, Klein et al., 1999, Klein, 2000a, Klein, 2000b, Bilbo and Nelson, 2001). Meadow voles of both sexes are photoperiodically induced breeders and, as such, when housed under a short light cycle (reproductively inhibitory) or in mixed-sex pairs under a long light cycle (reproductively stimulatory) express low or high levels of circulating gonadal hormones, respectively (Adams et al., 1980, Seabloom, 1985, Cohen-Parsons and Carter, 1987, Cohen-Parsons and Carter, 1988, Galea et al., 1995, Perrot-Sinal et al., 2000). As such, meadow voles represent an appropriate species to consider the effects of photoperiod and sex on LPS-induced changes in activity.

Accordingly, the present study assessed LPS-induced changes in locomotor activity in both male and female meadow voles housed under short or long light cycles. As well, the potential impact of photoperiod and sex on tolerance development were considered. Seven days after the initial injection, all voles were rechallenged with an identical treatment to assess the formation of behavioral tolerance to LPS. In addition, progesterone levels were assayed in a subset of female voles, as these levels have not been previously determined in our vole colony and to assess any potential impact of this hormone on sickness behaviors.

Locomotor activity levels were assessed 2 h after each injection using automated open-fields (Ossenkopp and Kavaliers, 1996), which have been used previously to quantify the effects of LPS on locomotor activity in male mice (Engeland et al., 2001b). This allowed for the simultaneous assessment of a variety of dissociable variables which, in the past, have been used to examine the effects of sex, age and other biological factors on locomotor activity in a variety of rodent species including meadow voles (e.g. Sanberg et al., 1985, Ossenkopp et al., 1987, Perrot-Sinal et al., 1998, Perrot-Sinal et al., 2000). Such a multivariate approach has been shown to provide an overall behavioral pattern which is more valid and reliable than any single measure of activity (Ossenkopp and Mazmanian, 1985, Ossenkopp et al., 1990). Moreover, the ecological validity of this multivariate approach has been confirmed in meadow voles, with wild caught meadow voles displaying increased activity levels with increasing levels of testosterone in a manner paralleling that of radio tracked animals in the field (Perrot-Sinal et al., 1998).

The winter immunoenhancement hypothesis (Nelson and Demas, 1996, Lochmiller and Moshkin, 1999, Nelson and Klein, 2000, Sinclair and Lochmiller, 2000) suggests that small mammals, which experience strong seasonal environmental fluctuations, should display increased immune responsiveness during the winter to better cope with the inherent stressors of this season such as food scarcity and increased thermoregulatory demands. Thus, increased immune functioning during the winter serves to counteract these immunosuppressive stressors and increases the chance of survival. Conversely, immune function is often compromised during the spring and summer, as there is then a transfer of metabolic energy from immunity to reproduction, so as to maximize reproductive output. Such tradeoffs of energy are an adaptive response to seasonal fluctuations and serve to increase the overall fitness of the organism. To date, laboratory studies of the effects of photoperiod on immune function have overwhelmingly reported an increase in immune functioning during short day lengths (Nelson et al., 1999). Thus, in the present study we hypothesize that immunocompetence should be greater in short day voles and, compared to long day voles, these animals should display less evident sickness following an initial injection of LPS and/or greater tolerance following a second injection of LPS.