Role of the glutamatergic system
Richard H. Melloni
John D. Coley, Marcelo Febo, Craig F. Ferris, James R. Stellar
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Arts and Sciences. Department of Psychology.
psychology, experimental, behavioral sciences, adolescent, aggression, anabolic steroids, glutamate, Syrian hamster, vasopressin
Anabolic steroids, Androgens, Aggressiveness in adolescence--Treatment
There is substantial evidence associating the use of anabolic androgenic steroids (AAS) during adolescence with the development of escalated levels of offensive aggression. In our laboratory we have used the Syrian hamster (Mesocricetus auratus) as an animal model to examine the neurobiological mechanisms underlying the development of aggression in adolescent AAS-treated animals (Melloni and Ricci, 2009). In this animal model the neural network modulating aggression is centered at the level of the anterior hypothalamus (AH), an area that shares reciprocal connections with other regions implicated in aggression control including the lateral septum (LS), bed nucleus of the stria terminalis (BNST), central amygdala (CeA), medial amygdala and the ventrolateral hypothalamus (VLH) (Delville et al., 2000). Preliminary studies showed that the activity of the aggression neural network is at least partially regulated by glutamate neurotransmission (Fischer et al., 2007). To date, however, the role of glutamate in the development of the aggressive phenotype in adolescent AAS-treated animals is unknown. The objective of this dissertation was to characterize the role of the glutamatergic system in adolescent AAS-induced offensive aggression. The first aim of this dissertation examined whether repeated adolescent AAS exposure altered glutamate expression in the center of aggression control, the AH. Immunohistochemical results showed that in the AH of aggressive AAS-treated animals there was a significant increase in the number of cells expressing phosphate activated glutaminase (i.e., PAG; the rate limiting enzyme for glutamate synthesis), vesicular glutamate transporter 2 (VGLUT2; an immunohistochemical marker for identification of glutamate cells) and PAG cells containing FOS (i.e., a marker of neuronal activation) (Carrillo et al., 2009; Carrillo et al., 2010a). Notably, close anatomical examination showed that changes in PAG, VGLUT2 and PAG/FOS were mainly localized to the ventrolateral portion of the AH (LAH). These findings supported a facilitatory role of glutamate in AAS-induced aggression and identified the LAH as a key subregion of the AH where AAS-induced changes in glutamate activity primarily occur. To further examine the temporal relationship between AAS-induced aggression and glutamate, the time course of AAS-induced neurodevelopmental and long-term effects on the glutamatergic system were investigated. Particularly, glutamate activity in brain areas part of the aggression circuit (i.e., the LAH, LS, BNST, CeA, MeA and VLH) was examined following 1, 2, 3, and 4 weeks of AAS treatment and following 1, 2, 3, and 4 weeks of withdrawal from AAS. Examination of the developmental time-course of AAS effects on aggression showed that the onset of aggression occurs following 2 weeks of exposure to AAS and continues to increase showing maximal aggression levels after 4 weeks of AAS-treatment. Moreover, this aggressive phenotype was detected after 2 weeks withdrawal from AAS. The time-course of AAS-induced changes in LAH-VGLUT2 closely paralleled increases in aggression. Increases in LAH-VGLUT2 were first detected in animals exposed to AAS for 2 weeks and were maintained up to 3 weeks cessation from AAS treatment. AAS treatment also produced developmental and long-term alterations in VGLUT2 expression within the BNST, CeA, MeA and VLH. However, the time course of AAS-induced changes within these regions was highly heterogeneous. Together, these data indicate that adolescent AAS treatment leads to alterations in the glutamatergic system in brain areas implicated in aggression control, yet only alterations in LAH-glutamate correlate with the time course of AAS-induced changes in the aggressive phenotype. Given that glutamate is the predominant excitatory neurotransmitter in the hypothalamus, that it modulates the majority of hypothalamic excitatory post-synaptic potentials and that AAS treatment induces increased activity of this neural system within the center of aggression control (i.e., the LAH), the next aim of this research determined whether in the LAH glutamate functioned as the aggression output system to other brain areas implicated in aggression control. This study used retrograde tracing to investigate glutamate specific alterations in the connections between the LAH and BNST, LS, MeA and VLH in AAS-treated animals. Specifically, animals were microinjected with retrograde tracer into the BNST (BNST group), LS (LS group), MeA (MeA group) or VLH (VLH group). Brains were then processed for VGLUT2 (i.e., BNST, LS and MeA) or PAG (i.e., VLH) immunofluorescence and examined for AAS-induced changes in 1) VGLUT2 or PAG, 2) cells with retrograde tracer and 3) VGLUT2 or PAG cells containing tracer within the LAH. Results showed that repeated adolescent AAS exposure produced significant increases in glutamate activity (i.e.,VGLUT2 or PAG) in all brain regions examined compared to sesame oil (SO)-treated controls. While no changes in the total number of cells with tracer were detected in the LAH of animals from the LS and MeA group, significant increases in the BNST group and decreases in the VLH group were observed. Moreover, when compared with SO-treated controls, animals from the BNST group showed a significant increase in the number of VGLUT2 immunopositive cells containing tracer within the LAH, while animals from the VLH and MeA group showed a significant reduction in the number of PAG and VGLUT2 cells containing tracer in the LAH. Lastly, no changes were detected in the number of VGLUT2 immunopositive cells with tracer within the LAH of AAS-treated animals in the LS group compared to vehicle-treated controls. Together these results indicate that glutamate likely functions as the aggression output system from the LAH and that adolescent AAS treatment significantly alters the neural circuitry modulating aggression. In addition, increases in the number of glutamate projections from the LAH to the BNST, identify this area as particularly important for the regulation of AAS-induced aggression and suggest that glutamate signaling within this area likely playskey role in the expression of aggression. Thus, to examine whether glutamate signaling within the BNST is necessary for the elevated aggressive response observed in AAS-treated animals, aggression levels were measured following administration of NBQX (i.e., an AMPA receptor antagonist, 0.5nmol, 1.0nmol or 2.0nmol) or saline. The results showed that in AAS-treated animals administration of NBQX into the BNST dose-dependently reduced aggressive behavior indicating, that glutamate signaling within the BNST is necessary for the expression of aggression in AAS-treated animals. Within the LAH, the activity of the glutamatergic system has been speculated to be under control of the neuropeptide vasopressin (AVP). Similar to glutamate, the activity of hypothalamic AVP has been heavily implicated in AAS-induced aggression. In fact, in Syrian hamsters, adolescent AAS treatment increases the availability of AVP and the afferent development of AVP neurons within the LAH (Harrison et al., 2000). Given that AVP-glutamate interactions have been shown to be critical for hypothalamic function (Bamshad and Albers, 1996; Chevaleyre et al., 2002) and that normal glutamate development is dependent on AVP (Chevaleyre et al., 2002), the next study investigated whether the stimulatory function of LAH-glutamate on aggression is dependent on AVP. Particularly this study examined whether in AAS-treated animals increased glutamate-specific connectivity between the LAH and BNST is dependent on the AVPergic system. In this investigate aggression levels in AAS-treated animals were measured following simultaneous administration of manning compound (i.e., an AVP V1a antagonist; 0.9mM) or saline into the LAH and AMPA (i.e., an AMPA receptor agonist; 0.01nm, 0.1nmol or 0.3nmol) or saline into the BNST. The results from this study replicated previous findings showing that blockade of LAH-AVP using an AVP V1a receptor antagonist significantly reduces aggressive behavior. Further, in hamsters administered the AVP V1a antagonist, stimulation of AMPA receptors within the BNST had a linear effect on aggression, where the smallest dose exacerbated the inhibitory effect of the AVP V1a antagonist, the medium dose had no effect and the highest dose recuperated aggression to control levels (i.e., AAS-treated animals microinjected saline into the BNST and LAH). Together, these data indicate that the activity of LAH-glutamate is dependent on AVP neurotransmission and that LAH-AVP signaling is necessary for the stimulatory effect of BNST-glutamate on aggression. Given these data we asked the question whether increased AVP-mediated stimulation of the glutamatergic system occurred due to AAS-induced increases in the number of AVP fibers innervating glutamate cells within the LAH. Notably, double-label immunofluorescence results showed a significant increase in the number of AVP fibers in apposition to VGLUT2 cells in the LAH of highly aggressive AAS-treated animals, compared to SO-treated controls. These data provide further evidence that support the importance of AVP-glutamate interactions in AAS-induced aggression and indicate that elevated AVP afferent innervation of glutamate cells within the LAH of aggressive AAS-treated animals is one of the neural mechanisms underlying increased AVP-dependent stimulation of the glutamatergic system within the LAH. In summary, the research from this dissertation provides compelling evidence supporting a key role of the glutamatergic system in the modulation of AAS-induced aggression, specifically having a facilitatory role in the expression of offensive aggression. In addition, short-term AAS exposure and long-term AAS withdrawal studies showed that AAS treatment produced alterations in glutamate activity in various brain areas implicated in aggression control however, only alterations in LAH-glutamate paralleled the time course of AAS-induced changes in aggressive behavior. Further, retrograde tracing and behavior pharmacology results identified the BNST as a critical brain area where the stimulatory activity of the glutamatergic system is necessary for the elevated aggression levels observed in AAS-treated animals. Lastly, the current research showed that synergistic stimulatory actions of AVP and glutamate are necessary for the expression of aggression in AAS-treated animals.
Carrillo, Maria, "Adolescent anabolic-androgenic steroids and the neurobiology of aggression: the role of the glutamatergic system" (2010). Psychology Dissertations. Paper 16. http://hdl.handle.net/2047/d20000283
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