Cranial irradiation induces axon initial segment dysfunction and neuronal injury in the prefrontal cortex and impairs hippocampal coupling

Abstract

Background: Radiation therapy for brain tumors commonly induces cognitive dysfunction. The prefrontal cortex (PFC) is crucial for a diverse array of cognitive processes, however, its role in radiation-induced cognitive dysfunction is unknown. We previously found that cranial irradiation impairs neuroplasticity along the hippocampal-PFC pathway. Herein, we hypothesized that brain irradiation directly affects the firing properties of PFC neurons, contributing to deficits in neuronal functions.

Methods: In vivo recordings were used to monitor the firing activities of PFC neurons and local field potentials in both PFC and hippocampal CA1/subicular regions after cranial irradiation of Sprague Dawley rats. We further assessed the impacts of irradiation on axon initial segments (AISs) with immunofluorescence assays of PFC slices.

Results: We found that PFC neurons exhibited increased excitation 3 days after radiation and the timing of increased excitation coincided with elongation of the AIS. At 2 weeks, excitation levels returned to nearly normal levels however the population of spontaneously firing neurons decreased. While the number of NeuN-positive neurons in the PFC was not different, persistent neuronal injury, manifested as ATF-3 staining, was present at 2 weeks. Radiation also disrupted communication along the hippocampal-PFC pathway, with elongation of the phase lag between regions. Analysis of paired-pulse ratios suggested that this was secondary to presynaptic dysfunction.

Conclusions: Cranial irradiation excited and injured surviving PFC neurons and was associated with a partial block of PFC's functional coupling to the hippocampus. These deficits in the PFC may contribute to radiation-induced cognitive dysfunction.

Keywords: PFC pathway; axon initial segments; cognitive impairment; cranial radiation; hippocampal; neuroplasticity.

Figure 1.

Whole-brain irradiation in a single 10-Gy dose transiently excited neurons in the prefrontal cortex (PFC). (A) A phase-contrast image illustrates that electrode general recording site within the PFC (arrow), (B) firing rates, and (C) numbers of spontaneously firing neurons per recording track were measured in adult rats at 3 and 14 days after radiation (RT). PFC neuron firing rates were 46.41 ± 12.50 Hz (n = 50 neurons, from 6 rats) at day 3, a significantly increase over rates in sham controls (Ctrl, 9.41 ± 1.69 Hz, n = 40 neurons, from 6 rats), but dropped to 9.64 ± 1.74 Hz (n = 33 neurons, from 6 rats) at day 14 after radiation [F_(2,120) = 6.204, P < .01]. The number of spontaneously firing neurons was initially increased (2.74 ± 0.26, n = 29 recording tracks, from 6 rats, day 3 after irradiation) and then reduced (0.97 ± 0.12, n = 36 recording tracks, from 6 rats, day 14 after irradiation), both significantly so relative those of sham controls (1.71 ± 0.28, n = 28 recording tracks, from 6 rats; [F_(2,90) = 15.56, P < .0001]). **P < .01 vs Ctrl; ^##P < .01 vs RT 3 days.

Figure 2.

Cranial irradiation altered the plasticity of the axon initial segment (AIS) of neurons in the prefrontal cortex (PFC). (A) Confocal imaging illustrates neuronal soma (green, anti-NeuN) and AIS (red, anti-Ank G) on a PFC slice (bar: 20 µm). (B) Histogram of AIS length showed a similar biphasic response in which the PFC neuron AIS was first elongated from 25.88 ± 0.31 (Ctrl, n = 110 from 6 rats) to 30.63 ± 0.45 at day 3 (n = 100 from 6 rats) and then reduced to 26.37 ± 0.27 at day 14 (n = 110 from 6 rats) after irradiation [F_(2,317) = 55.14, P < .0001]. **P < .01 vs Ctrl; ^##P < .01 vs RT 3 days.

Figure 3.

Cranial irradiation damaged, but did not reduce the number of, neurons in the prefrontal cortex (PFC). (A) Radiation did not significantly change the numbers of PFC NeuN-positive neurons (26.11 ± 0.83, n = 18 images from 6 rats, sham control; 24.61 ± 1.11, n = 18 images from 6 rats, day 3 after irradiation; 25.78 ± 1.07, n = 18 images from 6 rats, day 14 after irradiation [F_(2,51) = 0.61, P = .55]). (B) Immunohistochemical staining for ATF-3, a marker of neuronal injury, was detected in PFC neurons at (D) day 3 after irradiation and (E) at day 14 after irradiation, but (C) not in sham controls (0.55 ± 0.17, n = 18 images from 6 rats, sham control; 22.78 ± 1.27, n = 18 images from 6 rats, day 3 after irradiation; 24.78 ± 1.16, n = 18 images from 6 rats, day 14 after irradiation [F_(2,51) = 182.2, P < .0001]). **P < .01 vs Ctrl.

Figure 4.

Cranial irradiation partially blocked signal transmission between hippocampus and the prefrontal cortex (PFC). (A) Two 15-s segments of local field potentials recorded simultaneously showed synchronization between PFC and hippocampal activities from control and RT group. (B) Cross-correlations of the activities in the PFC and hippocampus were not significantly affected by cranial irradiation (0.31 ± 0.04, n = 40 neurons from 6 rats, sham control; 0.28 ± 0.03, n = 49 neurons from 6 rats, day 3 after irradiation; 0.32 ± 0.03, n = 33 neurons from 6 rats, day 14 after irradiation [F_(2,119) = 0.47, P = .636]). (C) Phase measurements, however, showed significant delays as early as day 3 that were still detectable at day 14 (6.33 ± 0.72 ms, n = 40 neurons from 6 rats, sham control; 30.43 ± 3.26 ms, n = 49 neurons from 6 rats, day 3 after irradiation; 13.43 ± 2.34 ms, n = 33 neurons from 6 rats, day 14 after irradiation [F_(2,119) = 25.86, P < .0001]), indicating that radiation notably blocked signal transmission between the PFC and hippocampus. **P < .01 vs Ctrl; ^##P < .01 vs RT 3 days.

Figure 5.

Cranial irradiation reduced the paired-pulse ratio and depressed Gamma EEG. (A) The paired-pulse ratio was significantly decreased at day 3 after irradiation in the prefrontal cortex (PFC) [F_(4,190) = 13.98, P < .0001]. Five animals were used in each group. (B) Spectrum powers at the 59–61 Hz frequency segment were significantly depressed in the PFC and hippocampus after 2 weeks after radiation. (PFC, Ctrl: 7.45E⁻³ ± 1.81E⁻³, n = 40 from 6 animals; RT 3 days: 1.74E⁻³ ± 0.20E⁻³, n = 50 from 6 animals; RT 2 weeks: 0.45E⁻³ ± 0.05E⁻³, n = 33 from 6 animals [F_(2,120) = 12.37, P < .0001]; Hippocampus, Ctrl: 6.38E⁻³ ± 1.15E⁻³, n = 40 from 6 animals; RT 3 days: 12.53E⁻³ ± 2.78E⁻³, n = 50 from 6 animals; RT 2 weeks: 0.29E⁻³ ± 0.07E⁻³, n = 33 from 6 animals [F_(2,120) = 8.64, P < .001]). **P < .01 vs Ctrl; ^##P < .01 vs RT 3 days.