Chronic inhibition of GABA synthesis in the bed nucleus of the stria terminalis elicits anxiety-like behavior

Abstract

The current study tested the hypothesis that chronic loss of inhibitory GABAergic tone in the bed nucleus of the stria terminalis (BNST), a region implicated in anxiety behavior, results in generalized anxiety disorder-like behaviors without panic-like responses (i.e., tachycardia, hypertension and tachypnea) following panicogenic stimuli (e.g., sodium lactate infusions). To test this hypothesis, the GABA synthesis inhibitor L-allylglycine (L-AG) or its inactive isomer D-AG was chronically infused into the BNST of male rats via osmotic mini-pumps. L-AG, but not D-AG, treated rats had increased anxiety-like behavior as measured by social interaction (SI) and elevated-plus maze paradigms. Restoring GABAergic tone, with 100pmoles/100nl of muscimol (a GABA(A) receptor agonist), in the BNST of L-AG treated rats attenuated L-AG-induced anxiety-like behavior in the SI test. To assess panic-like states, L-AG treated rats were intravenously infused with 0.5 M sodium lactate, a panicogenic agent, prior to assessing SI and cardiorespiratory responses. L-AG decreased SI duration again; however, sodium lactate did not induce panic-like cardiorespiratory responses. These findings demonstrate that GABA inhibition in the BNST elicits anxiety-like behavior without increasing sensitivity to lactate, thus suggesting a behavioral profile similar to that of generalized anxiety-like behavior rather than that of panic.

Figure 1

An illustration representing a timeline of experimental conditions (listed above arrows) from day 1 (D1) to day 26 (D26) along the horizontal bar. See methods for detailed description. Gray shading of horizontal bar indicates the timeline of D-AG or LAG infusions.

Figure 2

EPM test data from pre (day 6) and post (day 26) D-AG and L-AG infusions. A) The amount of time (sec) spent in the open arm of the elevated plus-maze or B) the number of entries into the open arm during a 5 min test of rats with previous infusions of the GABA synthesis inhibitor L-AG or its inactive isomer D-AG into the BNST. Bars represent the mean and error bars represent the SEM. *, p<0.05.

Figure 3

The amount of time (sec) that the experimental rats spent initiating interaction with the partner rats during a 5 min SI test, both before (day 5) and after (day 14) pump surgery. The top graph depicts the average times for animals implanted with L-AG pumps into the BNST, while the bottom graph shows times for those with D-AG pumps into the BNST. Bars represent the mean and error bars represent the SEM. *, p<0.05.

Figure 4

The amount of time (secs) that the experimental rats spent initiating interaction with the partner rats during a 5 min SI test, under four conditions. The first bar represents pre L-AG infusion baseline. Four days after L-AG infusion an SI test was done following an injection of saline (bar 2), 20 pmoles of muscimol (bar 3), or 100 pmoles of muscimol into the BNST. Treatments 2 – 4 were administered in a counter balanced design. Bars represent the mean and error bars represent the SEM. * Significantly different from pre-LAG baseline (paired t-test) and both doses of muscimol (Tukey’s posthoc test following ANOVA), # significantly different from 100 pmoles of muscimol (Tukey’s posthoc test following ANOVA). *,#, p<0.05.

Figure 5

Changes in A) social interaction time, B) heart rate, C) blood pressure and D) respiration rate in rats infused with i.v. sodium lactate prior to L-AG pump placement in the BNST, and on day 20 or 22 after beginning infusions with the GABA synthesis inhibitor L-AG and pre-injections of either saline or muscimol 100 pmoles into the BNST. There were no significant peripheral responses to lactate infusions in these animals either at baseline or after GABA synthesis inhibition in the BNST which resulted in increased baseline anxiety. In these animals, there was no difference in lactate responses after saline or muscimol pre-injections into the BNST, suggesting no differences in lactate sensitivity with or without baseline anxiety increases. * significant against pre L-AG in BNST (*, paired t-test, p<0.002) and L-AG in BNST + 100 pmoles muscimol (*, paired t-tests, p≤0.050). Bars represent the mean and error bars represent the SEM.

Figure 6

Distribution of L-allylglycine (L-AG: GABA synthesis inhibitor) or D-allylglycine (D-AG: control isomer) filled osmotic minipumps in the bed nucleus of the stria terminalus (BNST) region of all 6 D-AG treated rats and 4 of 7 L-AG treated rats. Placements were confirmed visually on 300 μm coronal brain section for the other 3 L-AG treated rats and then microdissected. A) Diagrammatic illustration of two coronal rat brain sections (−0.30 to −0.26 mm bregma and +0.20 to +0.48mm bregma) with minipump placement and treatment indicated by symbols (D-AG infusion site = black square and LAG infusion site = white circle) and boundaries of the BNST indicated by gray shading. B) Low magnification photograph of cresyl violet staining in the BNST region; the arrow denotes the minipump placement. Scale bar: 400 μm. Abbreviations: ac, anterior commissure; aca, anterior part of anterior commissure; cc, corpus collosum; CPu, caudate putamen; f, fornix; ic, internal capsule; LSD, dorsal part of lateral septum; LSI, intermediate part of lateral septum; LSV, ventral part of lateral septum; LV, lateral ventricle; MS, medial septum; VDB, nucleus of the vertical limb of the diagonal band.