The goal of the current study was to examine energetic trade-offs between reproduction and immune function in a seasonally breeding rodent, the Siberian hamster. A previous report indicated that short-day housed hamsters receiving daily injections of NMDA elevate circulating levels of LH and maintain breeding season-like gonads, while displaying a short-day-like decrease in body mass (Ebling et al., 1995
). Based on these findings, we predicted that NMDA would induce a dose-dependent rise in serum testosterone, and that, similar to the previous report, short-day housed hamsters receiving NMDA would maintain long day-like reproductive status while displaying the typical short-day reduction in energy reserves (e.g., reduced body fat and body mass). Indeed, daily NMDA injections provided to hamsters housed in short-day photoperiods displayed long-day-like gonadal morphology and short-day like reductions in body mass; short-day like reductions in fat pad mass was also observed in the current study. Further, short-day hamsters would have a reduced immune response compared with controls, owing to an energetic trade-off between reproduction and immune function; this predicted reduction in immunity, we predicted, would be driven by short-day induced decreases in energy reserves (e.g., fat stores). Contrary to these predictions however, NMDA treatment lead to greater antibody production in response to KLH, regardless of photoperiodic treatment. These findings suggest that NMDA enhances immune responses and that this immunoenhancement appears to be independent of changes in energetic state.
Mature reproductive organs are maintained by activation of the hypothalamo-pituitary-gonadal (HPG) axis. Seasonally breeding rodents typically experience a marked decline in reproductive function and thus reproductive success when exposed to winter-like short-days (Beery et al., 2007
). For example, hamsters transferred from long summer-like to short winter-like days display significant reductions in circulating levels of luteinizing hormone (LH) and testosterone, followed by subsequent involution of the testes, driven primarily by gonadal apoptosis (Maywood and Hastings, 1995
; Young et al., 1999
). Short-day induced reproductive suppression is orchestrated, in part, by changes in glutamatergic influences on hypothalamic GnRH neurons (Ebling et al., 1995
The current study sought to confirm that NMDA activates the HPG axis, and thus stimulates maintenance of a long day-like reproductive state in hamsters transferred to short days. Consistent with this hypothesis, injections of NMDA induced a dose-dependent rise in circulating testosterone titers in adult male Siberian hamsters, with the two highest doses (20 mg/kg and 40 mg/kg) eliciting similarly pronounced elevations in testosterone. This finding supports a previous report in this species, which demonstrated NMDA-induced up-regulation of pituitary LH release (Ebling et al., 1995
Next, to examine whether prolonged NMDA activation of the HPG axis maintains reproductive status in hamsters exposed to 8 weeks of short-day photoperiods, a period that typically leads to full gonadal regression and significant reductions in body mass and fat stores (Wade and Bartness, 1984
; Young et al., 1999
), hamsters received daily NMDA injections of 20 mg/kg for 8 weeks; this dose corresponds to the smallest dose from Experiment 1 that elicited a significant elevation in testosterone. Consistent with a previous report (Ebling et al., 1995
), the gonads of hamsters housed in short-day photoperiods that received daily NMDA injections were similar in size to those housed in long-day photoperiods. Interestingly, although these animals had long-day like reproductive organs, they exhibited a normal short-day like decrease in body mass (e.g. (Wade and Bartness, 1984
). Because photoperiodic changes in body mass are due in large part to changes in total body fat in this species (Wade and Bartness, 1984
), these findings suggest that NMDA did not alter total body fat. Indeed, short-day hamsters receiving NMDA or vehicle had similarly sized fat pads; these pads were considerably smaller than animals held in long-days. Thus, as expected, NMDA injections in short-day housed animals induced dissociations between energy reserves (e.g., fat) and reproductive function (e.g., gonads), suggesting that NMDA enables selective manipulation of the reproductive system without significant alterations in energetic state.
It has previously been demonstrated by our laboratory and others, that short-day hamsters exhibit a depressed antibody response to an immune challenge compared with long-day hamsters (Demas et al., 2002
; Drazen et al., 2001
; Yellon et al., 1999b
; Zysling and Demas, 2007
). This decreased antibody response occurs concomitantly with short-day induced decreases in energy stores, suggesting that decreased energy availability is driving the observed changes in immunity (Demas, 2004
). Thus, we hypothesized that experimentally increased investment in an energetically expensive physiological function, reproduction (experimentally activated via NMDA injections), coupled with the typical reductions in energy reserves in short-day hamsters, would lead to a trade-off in responses, thus eliciting a further suppression of antibody production. This outcome, however, was not observed; antibody responses in NMDA-injected hamsters displayed greater antibody titers in response to an immune challenge. These findings suggest a direct effect of NMDA on immune responses in this species.
Previous investigations have reported a significant decline in antibody responses in hamsters held on short versus long days (Demas, 2002
; Demas et al., 2002
; Drazen et al., 2001
; Drazen et al., 2000
; Yellon et al., 1999a
). However, in this investigation, no differences were observed between the vehicle-injected animals housed in differing photoperiods. The lack of clear photoperiod-induced changes in immune function in vehicle-treated animals may be due to the chronic mild stress imposed by daily injections. A recent report (Zysling and Demas, 2007
) observed that anti-KLH IgG levels animals receiving multiple injections of saline every other day over a 12 day did not differ compared with controls, consistent with the idea that multiple injections may indeed increase stress levels, reducing the ability to detect naturally occurring photo-induced reductions in immunity.
Within the central nervous system, the excitatory amino acid glutamate acts on distinct classes of receptors, which coordinate distinct physiological responses. NMDA acts on one such class of receptors, the so-called NMDA receptor subtype. NMDA glutamatergic receptors have recently been identified in the immune system on lymphocytes (Boldyrev et al., 2004
; Kostanyan et al., 1997
). These receptors have been shown to mediate the activation of T-cells and the potentiation of T-cell antibodies (Boldyrev et al., 2004
; Lombardi et al., 2001
; Miglio et al., 2005
). In the current study, increasing antibody production in response to NMDA injections may be possible through direct activation of the immune system through actions on T-cells. KLH, employed in the present study, is a T-cell dependent antigen; thus, antibody production requires coordination of both T and B lymphocytes (e.g. (Julius et al., 1972
). This idea of a direct effect of NMDA on antibody production is supported by our observation of elevated antibody responses in all animals receiving NMDA injections regardless of photoperiod; both long and short day NMDA-injected animals displayed significantly greater antibody production compared to animals receiving vehicle. Future studies will be needed to confirm this hypothesis. If true, greater antibody production in all NMDA treated hamsters regardless of photoperiod likely limited our ability to detect our hypothesized energetic trade-off between the reproductive and immune systems.
In addition to assessment of acquired immunity, the effects of daily NMDA injections on one aspect of the innate immune response were assessed by quantifying the ability of serum to kill E. coli
bacteria in vitro
. No significant differences were observed in serum bactericidal capacity; however, in short day NMDA injected animals, the mean percent of bacteria killed was slightly (~5%) higher, suggesting that NMDA may not only alter the acquired immune system, but may also play a minor mediating role in innate immune defenses. Consistent with this idea, NMDA activation increases intracellular reactive oxygen species (ROS) in lymphocytes (Boldyrev et al., 2004
), and ROS can act as key antimicrobial agents of the innate immune system (Bogdan et al., 2000
). Further work will be needed to address the potential role of NMDA in modulating innate immune system functioning.
In the present study, we utilized NMDA to dissociate reproductive and energetic responses to photoperiod. We hypothesized that this manipulation would provide a powerful way to investigate the cost of “mistimed” reproductive responses in seasonal mammals; the current study investigated this hypothesized cost in immune investment. The data from the current investigation suggest that this manipulation, while useful for manipulation reproductive function, should be used with caution when addressing complex physiological interactions. Future studies may, however be able to utilize this method to investigate other traits of behaviors associated with this uncoupling of physiological (large gonads) and morphological (pelage coloration and body mass) traits. For example, investigations of behavioral interactions between reproductive and non-reproductive individuals (e.g. (Carter et al., 1980
; Dluzen et al., 1981
) housed in the same photoperiod may yield interesting insights into the ability of a reproductive individual to alter the behavior or physiology of a non-manipulated animal. The current study employed peripheral injections of NMDA, which likely led to direct modulation of the immune system. Infusion of NMDA directly into the central nervous system may enable future studies to utilize this neurotransmitter to uncover potential indirect effects on the immune system associated with changes in HPG neuroendocrine activity.
The current results support a previous finding (Ebling et al., 1995
) demonstrating that the glutamate agonist NMDA blocks photoperiod induced gonadal regression, while leaving photoperiod-induced changes in energy intake or stores unaffected. We further documented a stimulatory effect of NMDA on antibody production, regardless of photoperiod. This observation of greater antibody production in animals treated daily with NMDA was unexpected and may demonstrate a potential key role for glutamate-signaling regulating interactions between the central nervous and the immune system. Indeed, evidence of interactions between the central nervous system and the immune system is becoming increasingly well-documented (for recent review see (Levite, 2001
). Collectively, these findings, combined with the known role of glutamate in the regulation of GnRH neurons (reviewed in (Brann and Mahesh, 1997
), suggest that glutamate, through actions on NMDA receptors may play a key modulatory role in regulating seasonal cycles of reproduction independent of photoperiodic-induced changes in adipocity, and that NMDA enhances humoral immunity via mechanisms that appear independent of changes in energy balance. Lastly, these data are consistent with the idea that the neurotransmitter glutamate may serve as a potential mechanism for communication between the central nervous and immune systems.