High Dose Gamma Radiation Selectively Reduces GABAA-slow Inhibition

Abstract

Studies on the effects of gamma radiation on brain tissue have produced markedly differing results, ranging from little effect to major pathology, following irradiation. The present study used control-matched animals to compare effects on a well characterized brain region following gamma irradiation. Male Sprague-Dawley rats were exposed to 60 Gy of whole brain gamma radiation and, after 24-hours, 48-hours, and one-week periods, hippocampal brain slices were isolated and measured for anatomical and physiological differences. There were no major changes observed in tissue appearance or evoked synaptic responses at any post-irradiation time point. However, exposure to 60 Gy of irradiation resulted in a small, but statistically significant (14% change; ANOVA p < 0.005; n = 9) reduction in synaptic inhibition seen at 100 ms, indicating a selective depression of the gamma-aminobutyric acid (GABA_A) slow form of inhibition. Population spike (PS) amplitudes also transiently declined by ~ 10% (p < 0.005; n = 9) when comparing the 24-hour group to sham group. Effects on PS amplitude recovered to baseline 48 hour and one week later. There were no obvious negative pathological effects; however, a subtle depression in circuit level inhibition was observed and provides evidence for 'radiomodulation' of brain circuits.

Keywords: brain slice; gaba; gamma; inhibition; pain therapy; radiomodulation; synapse; synaptic inhibition.

Figure 1. Drawing of a 400-µm thick rat hippocampal slice used for electrophysiological recordings.

Three major excitatory pathways of the hippocampal formation are depicted: the perforant pathway (pp) originating from the entorhinal cortex and projecting to granule cells of the dentate gyrus (DG), the mossy fiber pathway (mf) which is comprised of the granule cell axons extending to the pyramidal cells of the CA3, and finally the Schaffer collaterals (sc) make glutamate-mediated excitatory synapses with dendrites of the CA1 pyramidal cells and inhibitory interneurons. On the bottom, an enlarged view showing the relative positions of recording and stimulating electrodes in the area of interest: the CA1 region. Extracellular electrophysiological recordings are produced by stimulating Schaffer-collateral (sc) fibers using a bipolar tungsten microelectrode. Field potentials were recorded by placing a recording micropipette filled with ACSF at the border between stratum oriens and stratum pyramidale, near the axonal output side of the CA1 cell body layer.

Figure 2. Inhibitory circuitry of the CA1 region.

The panel on the left shows a schematic of the neuronal circuitry involved in the CA1 region of the hippocampus. The open circles represent glutaminergic, excitatory synapses and the closed darkened circles depict GABAergic, inhibitory synapses. Stimulation is sent in pulses, 100 ms apart (STIM) and recordings from different positions in the circuit exhibit different output responses. Recording from the dendritic (top) level presents as paired pulse potentiation (PPF), as a result of enhanced glutaminergic transmitter release on the second stimulus pulse. For the electrophysiological experiments discussed in this paper, a recording electrode was placed in the cell body region (bottom), as represented by the triangular shape labeled as CA1. Here, GABAA inhibition comes into play resulting in paired pulse inhibition (PPI) of the second population spike. The PPI ratio is calculated by dividing the amplitude of the first population spike by the amplitude of the second population spike.

Figure 3. Two forms of GABA inhibition can be pharmacologically isolated using selective antagonists for different types of GABA receptors.

Furosemide, a subunit-specific antagonist that exclusively blocks GABAA fast IPSCs changes the normal single population spike (PS) discharge into a pair of spikes, showing that GABAA fast inhibition comes on quickly (< 2 ms) but lasts only a short time (< 10 ms) and is responsible for limiting CA1 discharge to a single spike. Gabazine, an antagonist for GABAA fast and slow synaptic inhibition, results in multiple PSs lasting several hundred ms, showing that slow inhibition starts at ~ 5 ms and limits CA1 discharge over the time course studied in the present experiments (100 ms).

Figure 4. Synaptic responses comparing sham and irradiated recordings from brain slices.

Population spike recording obtained from a sham animal (left) and a recording from a slice harvested from a rat 24 hours after 60 Gy gamma radiation (right). Gamma radiation depressed GABAA slow inhibition as evidenced by the reduction in the PPI level. There was also a small decrease in the first pulse responses, with no evidence of a significant reduction of GABAA fast inhibition (no secondary PS response).

Figure 5. Gamma radiation of 60 Gy produced a small but statistically significant depression of GABAA slow-mediated inhibition 24 hours after irradiation that recovered after 48 hours and one week.

Amplitude of population spikes in mV for first PS (for CONTROL.1, 24 hr, 48 hr and one week slices) as well as second PS (for CONTROL.2 and 24 hr slices). Combined data shows a decrease in the first spike amplitude for 24 hr, 48 hr, and one week animals after irradiation. However, when compared to sham animal data, only 24 hr rats had a significant decrease (p = 0.002; ANOVA), while the decrease of first PS seen in 48 hr and one week animals were not significant. There was no further significant decrease of the first PS seen for 48 hr and one week animals when compared to animals observed 24 hr after radiation. Average ratios of first and second spikes to assess amount of inhibition via PPI values were determined for control, 24 hr and 48 hr rats (Bottom). A small but significant decrease (14.4% change; p = 0.035 ; n = 9) of PPI was noted in 24 hr animals when compared to control. The apparent continued decrease of PPI in 48 hr rats was not significant (p = 0.11). Error bars represent the standard deviation of the mean.

Figure 6. There was little or no apparent change in the overall anatomy of brain tissue in the CA1 region of the hippocampus (shown), or in any other brain region at the gross anatomy level.

Detailed histological analysis also showed little or no change in morphology, but immunohistochemistry revealed a small decrease in neuropeptide Y (NPY) expressing GABAergic interneurons. The number of NPY neurons present per section of the hippocampus in the CA1 region of sham animals (n = 6) and rats irradiated 24 hr (n = 6) prior to brain slice harvesting were analyzed. There was a significant decrease (p < 0.001) in the number of NPY positive cells in irradiated rats when compared to sham (CONTROL) animals.