The recovery of acetylcholinesterase activity and the progression of neuropathological and pathophysiological alterations in the rat basolateral amygdala after soman-induced status epilepticus: relation to anxiety-like behavior

Abstract

Organophosphorus nerve agents are powerful neurotoxins that irreversibly inhibit acetylcholinesterase (AChE) activity. One of the consequences of AChE inhibition is the generation of seizures and status epilepticus (SE), which cause brain damage, resulting in long-term neurological and behavioral deficits. Increased anxiety is the most common behavioral abnormality after nerve agent exposure. This is not surprising considering that the amygdala, and the basolateral nucleus of the amygdala (BLA) in particular, plays a central role in anxiety, and this structure suffers severe damage by nerve agent-induced seizures. In the present study, we exposed male rats to the nerve agent soman, at a dose that induce SE, and determined the time course of recovery of AChE activity, along with the progression of neuropathological and pathophysiological alterations in the BLA, during a 30-day period after exposure. Measurements were taken at 24 h, 7 days, 14 days, and 30 days after exposure, and at 14 and 30 days, anxiety-like behavior was also evaluated. We found that more than 90% of AChE is inhibited at the onset of SE, and AChE inhibition remains at this level 24 h later, in the BLA, as well as in the hippocampus, piriform cortex, and prelimbic cortex, which we analyzed for comparison. AChE activity recovered by day 7 in the BLA and day 14 in the other three regions. Significant neuronal loss and neurodegeneration were present in the BLA at 24 h and throughout the 30-day period. There was no significant loss of GABAergic interneurons in the BLA at 24 h post-exposure. However, by day 7, the number of GABAergic interneurons in the BLA was reduced, and at 14 and 30 days after soman, the ratio of GABAergic interneurons to the total number of neurons was lower compared to controls. Anxiety-like behavior in the open-field and the acoustic startle response tests was increased at 14 and 30 days post-exposure. Accompanying pathophysiological alterations in the BLA - studied in in vitro brain slices - included a reduction in the amplitude of field potentials evoked by stimulation of the external capsule, along with prolongation of their time course and an increase in the paired-pulse ratio. Long-term potentiation was impaired at 24 h, 7 days, and 14 days post-exposure. The loss of GABAergic interneurons in the BLA and the decreased interneuron to total number of neurons ratio may be the primary cause of the development of anxiety after nerve agent exposure.

Keywords: Acetylcholinesterase; Anxiety; Basolateral amygdala; Long-term potentiation; Soman; Status epilepticus.

Figure 1

Time course of the recovery of AChE activity after soman-induced status epilepticus (SE). AChE activity was reduced by more than 90% in all four brain regions at the onset of SE (n = 7), as well as at 24 hours later (n = 11). At 7 days post-exposure (n = 11), AChE activity had recovered in the BLA, while at 14 days (n = 10) and 30 days (n = 12) post-exposure, it was not significantly different from the control levels in all 4 brain regions. **p < 0.01; ***p < 0.001 compared to the controls (Welch F ANOVA with Games-Howell post hoc).

Figure 2

Time course of neuronal degeneration in the BLA after soman-induced SE. A, B. The BLA region where neuronal degeneration was assessed. In A, the drawings are from Paxinos and Watson (2005), while in B the BLA is outlined on FJ-C stained sections. C. Representative photomicrographs of FJC-stained sections, at 24-hours, 7-, 14-, and 30-days after soman exposure. Rows correspond to the coordinates shown in B. Total magnification is 20×. Scale bar, 250 μm. D. Quantitative assessment of the extent of neurodegeneration at 24-hours (n = 4), 7- (n = 6), 14- (n = 4), and 30 days (n = 5) after soman exposure. Values are mean ± SEM number of cells counted in the BLA from both hemispheres. **p < 0.01, ***p < 0.001 (One-Way ANOVA with Tukey post hoc).

Figure 3

Time course of neuronal loss in the BLA after soman-induced SE. A. Panoramic photomicrograph of a Nissl-stained section from half hemisphere, outlining the BLA where stereological analysis was performed (red highlight). B. Representative photomicrographs of Nissl-stained sections showing BLA cells from a control rat and from soman-exposed rats, analyzed at 24-hours, 7-, 14- and 30-days after exposure. Total magnification is 63× and scale bar is 50 μm. C. Group data of stereological estimation of the total number of Nissl-stained neurons in the BLA, expressed as percent of the control group (n = 11). Significant neuronal loss was present in rats at 24 hours (n = 5), 7 days (n = 6), 14 days (n = 5), and 30-days (n = 6) after soman exposure. There were no differences in the extent of neuronal loss among the 24-hour, 7-, 14-, and 30-day groups; ***p < 0.001 (One-Way ANOVA with Dunnett's T post hoc).

Figure 4

Delayed loss of GABAergic interneurons in the BLA after soman-induced SE. A. Panoramic photomicrograph of an immunohistochemically-stained section for GAD-67 (BLA is outlined in white). B. Representative photomicrographs of GABAergic interneurons in the BLA from a control rat and from soman-exposed rats, analyzed at 24-hours, 7-, 14- and 30-days after exposure. Total magnification is 630×; scale bar, 50 μm. C. Group data showing the mean and standard error of the stereologically estimated total number of GAD-67-positive cells in the BLA. D. Group data showing the mean and standard error of the ratio of GABAergic interneurons to the total number of neurons. *p < 0.05, **p < 0.01, ***p < 0.001 (Welch F ANOVA with Games-Howell post hoc and one-way ANOVA with Dunnett's T post hoc).

Figure 5

Exposure to soman causes long-lasting increases in anxiety-like behavior, as measured by the open field and acoustic startle response (ASR) tests. A. Soman-exposed rats spent significantly less time in the center of the open field, at 14 and 30 days after exposure, compared to control rats. B. Exposure to soman significantly increased the amplitude of the ASR to both the 110 and 120 dB startle stimuli, at 14 and 30 days after exposure, compared to controls. There were no significant differences between the 14- and 30-day groups. *p < 0.05, **p < 0.01; n = 17 (One-Way ANOVA with Dunnett's T post hoc).

Figure 6

Alterations in the BLA field potentials after soman-induced SE. A to E show representative field potentials evoked in the BLA by paired-pulse stimulation of the external capsule, from control rats (n = 9) and soman-exposed rats, at 24-hours (n = 9), 7 days (n = 9), 14 days (n = 10), and 30 days (n = 11) after exposure; each trace is an average of 10 to 15 sweeps. In the soman-exposed rats, higher stimulus intensities were necessary to evoke field potentials compared to controls, the duration of the field potentials was prolonged (notice the decay of the waveforms), and the paired-pulse ratio was significantly increased (F). **p < 0.01 (One-Way ANOVA with Dunnett's T post hoc).

Figure 7

Effects of soman exposure on Long-Term Potentiation in the BLA. The plots show the time course of the changes in the amplitude of the field potentials after high-frequency stimulation (HFS). The amplitude of the 3 responses recorded in each min (stimulation at 0.05 Hz) was averaged, and each data point on the plots is the mean and standard error of these averages, from 7 to 10 slices (see sample sizes in the text). Traces over the plots are examples from a control rat and from soman-exposed rats studied at the indicated time-point after exposure; the superimposed field potentials are a baseline response and a response at 50 to 60 min after HFS (each trace is the average of 10 to 15 sweeps). Potentiation of the responses, measured at 50 to 60 min after HFS, was significantly lower – compared to the control group – at 24 hours, 7 days and 14 days post-exposure, but not at 30 days post-exposure.