In Experiment 1
, LPS injection resulted in high levels of crouching, eye-closing, and piloerection, the same behaviors seen in guinea pig pups during protracted (3 hr) periods of isolation. This finding replicates previous results (Hennessy et al., 2004
), and confirms that the passive behaviors characteristic of prolonged isolation are the same behaviors associated with unequivocal sickness. In Experiment 1
, it was also found that ICV infusion of 25 μg of α-MSH significantly reduced the effect of LPS on two of the passive responses, crouching and piloerection. Because this is the same dose and route of administration previously used to reduce passive behaviors during protracted isolation (Schiml-Webb et al., 2006
), the results support the conclusion that α-MSH's effect in the previous study was due to it anti-inflammatory properties, rather than some other potential mode of action.
The most unexpected result was the suppressive effect of α-MSH on active behavior in Experiment 1
. Under saline-injected control conditions, vocalizing was significantly reduced, and line-crossing was reduced to a marginally significant level. Based on past results (Schiml-Webb et al., 2006
, and unpublished findings), we had not predicted a significant effect of α-MSH on the active behaviors, though if we had, we would have predicted an enhancement rather than a suppression of these behaviors for either of two reasons. First, a reduction in active behavior is a commonly observed sickness response (Hart, 1988
) and so an anti-inflammatory might be expected to oppose this effect and increase activity. Second, the vocalizing of guinea pig pups during isolation procedures is considered to reflect an anxiety-like state (Borsini, Podhorna, & Marazziti, 2002
). Since α-MSH has been found to produce anxiogenic effects in some species (Panksepp & Abbott, 1990
; Rao et al., 2003
) there would be reason to expect α-MSH would increase vocalizing. It may be worth noting in this regard that the subjective impression of the primary observer during Experiment 1
was that α-MSH had a calming
effect on the pups. At present, we see no satisfying explanation for the effects of α-MSH on active behavior in the saline-inject pups of Experiment 1
. Clearly, this aspect of the findings will require more investigation to fully understand. We can say, however, that the present results argue against ICV α-MSH having an anxiogenic action in the young guinea pig.
For active behaviors, there also was a marginally significant tendency for α-MSH to reduce the effect of LPS in Experiment 1
. However, this tendency may have been an artifact. As just noted, a-CSF and α-MSH-infused pups exhibited rather different levels of active behaviors when injected with saline. Further, LPS greatly suppressed behavior in both groups. (Note the similarity of median values of behavior for the “saline” and “saline minus LPS” scores in .) Thus, the tendency for a-CSF and α-MSH groups to differ on the “saline minus LPS” measure was likely due to the differential values in the saline-injected control condition, perhaps combined with a “floor” effect in the suppressive action of LPS.
In Experiment 2
, indomethacin significantly reduced the crouching and full passive response of guinea pig pups over the course of a prolonged (i.e., 3-hr) isolation in a novel cage. Active behaviors were not affected. Since indomethacin disrupts synthesis of prostaglandins, a final common mediator of many inflammatory effects, but has no obvious other means of reducing passive behavioral responses, the results of Experiment 2
further implicate proinflammatory factors in the expression of the passive responses during protracted isolation.
α-MSH and indomethacin impair inflammation through distinct processes. The effects of α-MSH are extremely broad and involve multiple modes of action. This peptide acts on an assortment of immune cells (monocytes, macrophages, dendritic cells, glia) in both the periphery and CNS to downregulate expression of various proinflammatory cytokines and increase production of the anti-inflammatory cytokine IL-10, as well as on CNS receptors to produce other anti-inflammatory neural effects (Lipton et al., 1999
; Lugar et al., 2004). Indomethacin's effects are more circumscribed, inhibiting synthesis of cyclooxygenase, the rate limiting factor in the production of prostaglandins. In the brain, prostaglandins are necessary mediators of some actions of proinflammatory cytokines (Rivest, 1999
), including a subset (Avitsur, Weidenfeld, & Yirmiya, 1999; Dunn & Swiergiel, 2000
; Johnson, Curtis, Dantzer, & Kelley, 1993
; Yirmiya, Barak, Avitsur, Gallily, & Weidenfeld, 1997
), though not all (Deak, D'Agonstino, Bellamy, Rosanoff, McElderry, & Bordner, 2005
) behavioral components of sickness. Thus, the effect of indomethacin in Experiment 2
indicates a contribution of prostaglandins to the passive behaviors of guinea pig pups. Moreover, the combined results of the two experiments provide convergent evidence supporting the hypothesis (Hennessy et al., 2001
) that the passive responses of guinea pig pups during a several hour period of isolation in a novel environment are mediated by proinflammatory factors, and therefore, examples of stress-induced sickness behaviors.
The stress-responsive peptide corticotropin-releasing factor (CRF) provides one potential means by which the isolation procedure may increase proinflammatory activity. CRF is known to stimulate various proinflammatory responses (Leu & Singh, 1992
; Singh, Pang, Alexacos, Letourneau, & Theoharides, 1999
; Webster et al., 1996
). Further, injection of CRF will increase passive responses of isolated guinea pig pups during a brief isolation much as was observed for LPS in Experiment 1
of the present report (Hennessy et al., 1995
). Since a CRF antagonist can delay the onset of the passive responses in isolated pups (McInturf & Hennessy, 1996
), it appears that endogenous CRF may be in part responsible for the transition from the initial active, to the subsequent passive, phase of responsiveness. Recently we found that α-MSH attenuates the passive responses of guinea pig pups injected with CRF (Schiml-Webb, Miller, Deak, & Hennessy, in preparation
), indicating that CRF acts through a proinflammatory mechanism. Together, these findings suggest that CRF release during the stress of isolation in a novel environment stimulates proinflammatory activity which promotes the passive responses (Hennessy, Deak, Schiml-Webb, & Barnum, in press
). Moreover, evidence that proinflammatory cytokines can, in turn, stimulate CRF release (Dunn, 2005
), suggests the possibility of positive feedback from cytokines to CRF to further amplify behavioral effects.
Recently, proinflammatory factors have been implicated in human depression. The so-called “cytokine hypothesis” holds that increased proinflammatory cytokines underlie some forms of depressive illness, and that in these cases, sickness responses contribute to behavioral symptoms (Schiepers, Wichers, & Maes, 2005
). We believe this hypothesis may be of particular interest in the context of maternal separation studies. In human infants, the “despair” stage was at the outset considered a form of depression (Spitz, 1946
), and this stage in nonhuman primates traditionally has been viewed as an animal model for depression (McKinney, Moran, & Kraemer, 1984
; Willner, 1991
). While it remains unclear if the passive responses of separated primates can be regarded as sickness behaviors, and though caution is required in generalizing from studies of sickness in laboratory animals to depression in humans (Dunn, Swiergiel, & de Beaurepaire, 2005
), the possibility that prolonged separation can lead to depressive-like symptoms through proinflammatory processes suggests a new framework in which these early findings might be considered.