Immunoreactivity of Muscarinic Acetylcholine M2 and Serotonin 5-HT2B Receptors, Norepinephrine Transporter and Kir Channels in a Model of Epilepsy

Abstract

Epilepsy is characterized by an imbalance in neurotransmitter activity; an increased excitatory to an inhibitory activity. Acetylcholine (ACh), serotonin, and norepinephrine (NE) may modulate neural activity via several mechanisms, mainly through its receptors/transporter activity and alterations in the extracellular potassium (K⁺) concentration via K⁺ ion channels. Seizures may disrupt the regulation of inwardly rectifying K⁺ (Kir) channels and alter the receptor/transporter activity. However, there are limited data present on the immunoreactivity pattern of these neurotransmitter receptors/transporters and K⁺ channels in chronic models of epilepsy, which therefore was the aim of this study. Changes in the immunoreactivity of epileptogenesis-related neurotransmitter receptors/transporters (M2, 5-HT2B, and NE transporter) as well as Kir channels (Kir3.1 and Kir6.2) were determined in the cortex, hippocampus and medulla of adult Wistar rats by utilizing a Pentylenetetrazol (PTZ)-kindling chronic epilepsy model. Increased immunoreactivity of the NE transporter, M2, and 5-HT2B receptors was witnessed in the cortex and medulla. While the immunoreactivity of the 5-HT2B receptor was found increased in the cortex and medulla, it was decreased in the hippocampus, with no changes observed in the M2 receptor in this region. Kir3.1 and Kir6.2 staining showed increase immunoreactivity in the cerebral cortex, but channel contrasting findings in the hippocampus and medulla. Our results suggest that seizure kindling may result in significant changes in the neurotransmitter system which may contribute or propagate to future epileptogenesis, brain damage and potentially towards sudden unexpected death in epilepsy (SUDEP). Further studies on the pathogenic role of these changes in neurotransmitter receptors/transporters and K⁺ channel immunoreactivity may identify newer possible targets to treat seizures or prevent epilepsy-related comorbidities.

Keywords: 5-HT2B; M2; PTZ; chronic epilepsy; norepinephrine.

Figure 1

Pictorial representation depicting the current experiment. This figure was created with BioRender.com (accessed on 7 March 2021). Ach, Acetylcholine; Kir, inwardly rectifying K⁺; NE, norepinephrine; PTZ, Pentylenetetrazol

Figure 2

Racine score exhibited by sham control and PTZ-kindled epilepsy rats. Values are presented as the mean ± SEM, n = 10 for each group, *** p < 0.001.

Figure 3

Body weight changes in control (A) and PTZ-kindled epilepsy (B) groups between day 1 and day 28 of treatment, n = 10 for each group, p > 0.05.

Figure 4

Haematoxylin and Eosin (H&E) staining images of the brain (cortex, hippocampus, and medulla) sections stained with purple in control (A–C, respectively) and PTZ-kindled epilepsy (D–F, respectively) groups. The black, yellow, and red arrows denote necrotic cells, cell degeneration, and haemorrhagic areas, respectively. Magnification of ×200. Scale bar: 50 μm.

Figure 5

Representative images of M2 receptors within the cortex, hippocampus, and medulla in control (A–C, respectively) and PTZ-kindled epilepsy of rat model (D–F, respectively). Figure (G) represents the M2 ACh receptor’s immunoreactivity in the cortex, medulla, and hippocampus regions. Values are presented as the mean ± SEM, n = 10, ** p < 0.01 and *** p < 0.001. The black arrow indicates the increased immunoreactive cells. Pictures were taken at a magnification of ×200. Scale bar: 50 µm.

Figure 6

Representative images of immunoreactivity for serotonin receptors within the cortex, hippocampus, and medulla in control (A–C, respectively) and PTZ-kindled (D–F, respectively). Figure (G) represents the immunoreactivity of the serotonin receptor 2B in several regions (cortex, medulla, and hippocampus). Values are presented as the mean ± SEM, n = 10, * p < 0.05, and *** p < 0.001. The black arrow indicates the immunoreactive cells. Pictures were taken at a magnification of ×200. Scale bar: 50 µm.

Figure 7

Representative images of immunoreactivity for NE transporter within the cortex, hippocampus, and medulla in control (A–C, respectively) and PTZ-kindled (D–F, respectively). Figure (G) represents the immunoreactivity of the NE transporter in several regions (cortex, medulla, and hippocampus). Values are presented as the mean ± SEM, n = 10, * p < 0.05, ** p < 0.01 and *** p < 0.001. The black arrow indicates the increased immunoreactive cells. Pictures were taken at a magnification of ×200. Scale bar: 50 µm.

Figure 8

Representative images of immunoreactivity for Kir3.1 channel within the cortex, hippocampus, and medulla in control (A–C) and PTZ-kindled epilepsy of rat model (D–F), respectively. Figure (G) represents the immunoreactivity of the Kir3.1 channel in several regions (cortex, medulla, and hippocampus). Values are presented as the mean ± SEM, n = 10, ** p < 0.01 and *** p < 0.001. The black arrow indicates the changed immunoreactive cells. Pictures were taken at a magnification of ×200. Scale bar: 50 µm.

Figure 9

Representative images of immunoreactivity for Kir6.2 channel within the cortex, hippocampus, and medulla in control (A–C) and PTZ-kindled epilepsy of rat model (D–F), respectively. Figure (G) represents the immunoreactivity of the Kir6.2 channel in several regions (cortex, medulla, and hippocampus). Values are presented as the mean ± SEM, n = 10, * p < 0.05 and *** p < 0.001. The black arrow indicates the changed immunoreactive cells. Pictures were taken at a magnification of ×200. Scale bar: 50 µm.