The present study demonstrates that a decrease in GABA inhibition due to chronic infusion of L-AG but not D-AG, the inactive isomer, into the BNST increases anxiety-like behaviors as measured by decreases in the amount of time spent in the open arms in the elevated plus-maze, as well as reductions in the duration of social interactions. These results are consistent with the idea that increased activation of the BNST is associated with the expression of anxiety-like responses. In the current study we assessed animals in the elevated plus-maze before and following treatment. Previously, File and colleagues demonstrated a change in benzodiazepine response on the 2nd
trial of the maze (File and Zangrossi, 1993
; File et al., 1993
), suggesting that on the 2nd
trial the response is confounded by 1 trial learning and thus different than that in the 1st
test. However, the response to benzodiazepines is re-established after 2 weeks of no further exposure to EPM. As a result, we assess our animals in the 2nd
trial at least 2 weeks from the first session.
Another important finding is that L-AG infusion into the BNST of the rats increased anxiety-like behavior without eliciting panic-like symptoms (i.e., tachycardia, tachypnea and increased blood pressure) in response to sodium lactate. This lack of responsivity is in contrast to that seen in our other animal models of anxiety. For example, similar L-AG infusions into the DMH produces anxiety-like behaviors in SI, but also makes the animals susceptible to displaying panic-like characteristics (i.e., tachycardia, tachypnea and increases in blood pressure) in response to i.v. infusions of 0.5M sodium lactate (Shekhar et al., 1996
; Johnson and Shekhar, 2006
; Shekhar et al., 2006
). Overall, this data supports the hypothesis that BNST plays an important role in regulating general anxiety. More importantly, these findings show that removing GABAergic tone in the BNST may be a useful tool in studying general anxiety-related circuits.
The data presented here are also consistent with results from other studies. Singewald et al. (2003)
found that systemic administration of four different anxiogenic compounds consistently increased cellular responses (i.e., c-Fos expression) in the BNST, while Lee and Davis (1997)
have shown that directly activating the BSNT with the stress associated neuropeptide corticotrophin releasing factor (CRF) will enhance acoustic startle responses (For review see Bale and Vale, 2004
). Interestingly, activation of the BNST with CRF will also induce reinstatement of cocaine use similar to footshock stress, while the CRF receptor antagonist D-Phe CRF will block reinstatement following the stress (Erb and Stewart, 1999
). The current findings together with the other behavioral studies strongly indicate that activation of the BNST is associated with increases in anxiety-like behaviors.
In addition to regulating anxiety-associated behavior, the BNST appears to be important in regulating social behavior. Studies conducted by Insel and colleagues have focused on social behavior in rats and prairie voles and found that oxytocin is a key neurotransmitter in inducing prosocial behaviors and have proposed that oxytocin may elicit prosocial behaviors through interactions with oxytocin receptors in the BNST (Insel, 1992
). In addition, recent studies indicate that the administration of the CRF receptor antagonist D-Phe CRF into the BNST blocks the submissive-defensive behaviors of hamsters normally associated with social defeat (Jasnow et al., 2004
). These results suggest that the BNST is also an important limbic structure for the expression of appropriate social behaviors across several species.
Animals in the L-AG treatment group demonstrated a decrease in social interaction time and open arm time, thus it could be suggested that L-AG infusions into the BNST may be reducing overall locomotor activity. However, there was not a significant difference between the L-AG and the D-AG treatment groups in the total number of entries (data not shown), therefore it would suggest that locomotion was not a confounding factor.
In order to confirm that L-AG infusions were inducing anxiety states by removing local GABAergic tone in the BNST and not other possible non-specific effects, local GABAergic tone was restored with acute injections of muscimol (a GABAA
receptor agonist) into the BNST. Injecting the highest dose of muscimol into the BNST of L-AG treated rats attenuated L-AG-induced anxiety responses in two separate experiments. Another possible confound would be diffusion of the L-AG into other nuclei. Earlier studies characterizing site specificity of L-AG infusions with exactly the same type of pumps in another brain area (the DMH) showed no significant effect in the adjacent areas beyond about one mm of radius when studies by either immunocytochemical or biochemical methods (Johnson and Shekhar, 2006
; Shekhar et al., 2006
), suggesting a limited area of diffusion from the pumps. Overall, these data are consistent with the hypotheses that L-AG infusions in the BNST elicits anxiety by removing local GABAergic tone and also suggests that the anxiolytic effects of GABA in the BNST are mediated via the GABAA
receptor. Furthermore, the chronic anxiogenic-like effects of GABA inhibition (i.e., L-AG treated) in the BNST suggests that GABA is tonically inhibiting the BNST. Intracellular recordings within the dorsal oval nucleus of the BNST, the area targeted for the pumps, reveals that some of the local GABAergic neurons are tonically active and that administration of bicuculline methiodide (BMI, a GABAA
receptor antagonist) attenuates this tonic inhibition (Rainnie, 1999
; bEgli and Winder, 2003
). Interestingly, rats undergoing ethanol withdrawal display marked anxiety-like behavior, which coincides with robust increases in CRF levels in the BNST (Olive et al., 2002
). If alcohol is given to these animals CRF levels in the BNST return to normal. However, if they are only given a regular diet, the levels will continue to increase 100% above baseline. This phenomenon may explain the anxiety associated with withdrawal as well as the increase in socialization following alcohol consumption.
Previous work in our laboratory has shown that an acute decrease in GABA functioning in the BLA or the DMH of rats will also elicit anxiety-like responses (Shekhar et al., 1990
; Sanders and Shekhar, 1991
; Shekhar, 1993
; Shekhar et al., 1993
; Sanders and Shekhar, 1995
; Shekhar and Katner, 1995
; Shekhar et al., 1996
; Sajdyk and Shekhar, 1997
). When we induced chronic loss of GABAergic inhibition in these regions of rats, either via L-AG infusions in the DMH or repeated subthreshold doses of BMI into the BLA, the animals display increased anxiety-related behavior and develop a sensitivity to i.v. sodium lactate similar to that of individuals with panic disorder [i.e., tachycardia, hypertension and tachypnea (Shekhar et al., 1996
; Shekhar and Keim, 1997
; Sajdyk and Shekhar, 2000
; Shekhar and Keim, 2000
Intravenous sodium lactate infusions have been best studied in subjects with panic disorder and consistently elicit panic attacks in the majority of panic disorder patients, often similar to naturally occurring episodes (Gorman et al., 1983
; Carr et al., 1986
; Gaffney et al., 1988
; Den Boer et al., 1989
; Hollander et al., 1989
; Yeragani et al., 1989
). Although panic-like response to lactate infusions is most consistent in panic disorder, some sensitivity to lactate infusions have been reported in post-traumatic stress disorder, where it primarily induces flash-backs (Rainey et al., 1987
; Jensen et al., 1997
), and a subset of patients with premenstrual syndrome (Facchinetti et al., 1992
; Sandberg et al., 1993
). A disorder that also shares a biological linkage with panic disorder (Facchinetti et al., 1998
). In contrast, lactate infusions are ineffective in patients with obsessive compulsive disorder (Gorman et al., 1985
), social anxiety disorder (Liebowitz et al., 1985
) and major depression without history of panic attacks (Cowley et al., 1987
). Thus, lactate induced panic has been proposed as a reasonable biological probe to separate panic versus other anxiety disorders (Cowley and Arana, 1990
). The lack of responsiveness to sodium lactate in the present study suggests that the anxiety-like behavior displayed by the rats with the L-AG pumps placed in the BNST is not like that of panic and may be more similar to that of generalized or social anxiety disorder.
The likelihood of BNST mechanisms being a possible substrate for generalized anxiety disorders is further supported by studies with the administration of buspirone, a 5-HT1A
agonist, into the BNST. Buspirone blocks light-enhanced startle (Davis et al., 1997
). It has also been shown that a natural stressor, such as chronic restraint, increases dendritic remodeling and arborization in the BNST, indicating a function in stressed-induced facilitation of anxiety (Vyas et al., 2003
). Furthermore, electrophysiological studies in the BNST also support a GABAergic and serotonergic mechanisms for reducing activation (Rainnie, 1999
; bEgli and Winder, 2003
) and thus reduce anxiety.