Neurotransmitter synthesis in newborn neurons in adult rat cerebral cortex after photothrombotic ring stroke with spontaneous reperfusion

Abstract

Under physiological conditions, neurons are not regenerated in the cerebral cortex in most mammalian species in the adult brain. However, neurogenesis occurs in the cerebral cortex in adult rats exposed to photothrombotic ring stroke 1 and in the penumbral cortex and ipsilateral striatum in adult rats after middle cerebral artery suture occlusion 2. The functional status of the post stroke cortical neurogenesis is unknown. In our previous studies, newborn neurons were seen to extend neurite like structures, which suggests that these cells might be potentially functional in the post stroke cortex and striatum. Whether or not the newborn neurons may synthesize neurotransmitters is unknown. The present study aimed to explore possible neurotransmitter synthesis during post stroke cortical neurogenesis. The adult male wistar rats, weighing 280–320 g, were used for the induction of photothrombotic ring stroke with spontaneous reperfusion and morphological recovery in the cortical region at risk 3. The DNA duplication marker BrdU was intraperitoneally injected (10 mg/kg body weight) repeatedly, which ended at day 7th post stroke. The rats were sacrificed at day 2nd, day 7th and day 30th post stroke. Coronal brain sections were processed for single/double immunohistochemistry and single/double immunofluorescent cell labeling. To identify the newborn cells, mouse anti-BrdU was used. To detect the neurotransmitters in cortical neurons, rabbit anti-Ach, GABA and glutamate were chosen. To examine the acetylcholine synthesizing enzyme, rabbit anti-ChAT (choline acetyl transferase) was used. DAPI was used to counterstain the nuclear DNA. The double/triple immunofluorescently labeled brain sections were analyzed by 3-D confocal microscope. Numerous BrdU immunolabeled cells appeared in the cortical region at risk at post stroke day 2nd, day 7th and day 30th. Under double immunohistochemistry, some of the cortical cells immunolabeled by BrdU in the nuclei were double labeled by Ach, GABA, glutamate or ChAT in the cytoplasm as examined under high magnification light microscope. The double labeled cells were randomly distributed in the cortical layer II to VI, more in the region at risk than in the adjacent cortex. Under 3-D confocal analysis, the BrdU immunolabeled cell nuclei were colocalized with Ach, GABA, glutamate or ChAT in the same cortical cells at various times post stroke. This study suggests that newborn neurons in the post stroke cortex were capable of synthesizing Ach, GABA and glutamate, a process which is of fundamental importance for the new neurons to function in the post stroke adult brains.