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. 2023 May 22;2(6):pgad166.
doi: 10.1093/pnasnexus/pgad166. eCollection 2023 Jun.

Administration of the glutamate-modulating drug, riluzole, after stress prevents its delayed effects on the amygdala in male rats

Siddhartha Datta  1 , Zubin Rashid  1 , Saptarnab Naskar  2 , Sumantra Chattarji  1
Affiliations

Administration of the glutamate-modulating drug, riluzole, after stress prevents its delayed effects on the amygdala in male rats

Siddhartha Datta et al. PNAS Nexus. .

Abstract

Extracellular glutamate levels are elevated across brain regions immediately after stress. Despite sharing common features in their genesis, the patterns of stress-induced plasticity that eventually take shape are strikingly different between these brain areas. While stress causes structural and functional deficits in the hippocampus, it has the opposite effect on the amygdala. Riluzole, an FDA-approved drug known to modulate glutamate release and facilitate glutamate clearance, prevents stress-induced deficits in the hippocampus. But whether the same drug is also effective in countering the opposite effects of stress in the amygdala remains unexplored. We addressed this question by using a rat model wherein even a single 2-h acute immobilization stress causes a delayed expression of anxiety-like behavior, 10 days later, alongside stronger excitatory synaptic connectivity in the basolateral amygdala (BLA). This temporal profile-several days separating the acute stressor and its delayed impact-allowed us to test if these effects can be prevented by administering riluzole in drinking water after acute stress. Poststress riluzole not only prevented the delayed increase in anxiety-like behavior on the elevated plus maze but also blocked the increase in spine density on BLA neurons 10 days later. Further, stress-induced increase in the frequency of miniature excitatory postsynaptic currents recorded in BLA slices, 10 days later, was also blocked by the same poststress riluzole administration. Together, these findings underscore the importance of therapeutic strategies, aimed at glutamate uptake and modulation, in correcting the delayed behavioral, physiological, and morphological effects of stress on the amygdala.

Keywords: acute immobilization stress; anxiety; excitatory synapses; riluzole; spine plasticity.

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Figures

Fig. 1.
Fig. 1.
Riluzole in drinking water after stress prevents the delayed increase in anxiety-like behavior. A) Experimental design depicting the timeline of single episode of immobilization stress (2 h), beginning at 10 AM on day 0. Riluzole is administered on day 0 after the end of the 2-h stress (12 PM) and continued for 24 h into day 1. On day 1 (12 PM), the drug is replaced by regular drinking water (vehicle) and continued till day 10. On day 10, anxiety-like behavior was assessed using EPM. Summary graph showing percentage of time spent in open arm B), percentage of entries into open arm C) on the elevated plus maze, and overall anxiety index D) on day 10. Two-way ANOVA, post hoc Tukey's multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. †P < 0.05 and ††P < 0.001 in “interaction” between factors “stress’ and “riluzole.”
Fig. 2.
Fig. 2.
Riluzole in drinking water after stress prevents the delayed increase in spine density on BLA principal neurons. A) Experimental design. Rats were subjected to a 2-h stress followed by 24-h riluzole treatment on day 0. Iontophoretic dye labeling carried out on individual BLA principal neurons on day 10. B) Spines were quantified only on the primary dendrites emanating from the main shaft of the principal neuron. C) Representative confocal images of dendritic segments and spines from BLA neurons in vehicle- and riluzole-treated animals (scale = 10 μm). D) Summary bar graph showing total number of spines in 80 μm length of primary dendrite. Ordinary two-way ANOVA, post hoc Tukey's multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ††P < 0.01 in “interaction” between factors stress and riluzole. E) Plot showing number of spines in successive 10-μm segments along the length of a primary dendrite. Repeated measures two-way ANOVA, post hoc Tukey's multiple comparisons test for “stress” versus “stress + riluzole.” ***P < 0.001 and ****P < 0.0001.
Fig. 3.
Fig. 3.
Poststress riluzole administration in drinking water prevents the delayed increase in frequency of mEPSCs on BLA principal neurons. A) Experimental design. Rats were subjected to a 2-h stress followed by 24-h riluzole treatment on day 0. Whole-cell patch clamp recordings of mEPSCs were carried out on BLA principal neurons on day 10. B) Representative traces of mEPSCs from BLA principal neurons in vehicle- and riluzole-treated animals (scale: 20 pA, 0.5 s). C) Summary graph showing frequency of mEPSCs on day 10. Two-way ANOVA, post hoc Sidak's multiple comparisons test, *P < 0.05 and **P < 0.01. D) Cumulative frequency distribution of mEPSCs in vehicle- and riluzole-treated groups. E) Graph showing average amplitude of mEPSCs in all groups.
Fig. 4.
Fig. 4.
Summary. Acute stress (top) causes a delayed increase in anxiety-like behavior, BLA spine density, and mEPSC frequency. These behavioral and synaptic changes are blocked by poststress riluzole treatment (bottom).
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