Hypoxic Preconditioning Differentially Affects GABAergic and Glutamatergic Neuronal Cells in the Injured Cerebellum of the Neonatal Rat

Abstract

In this study we examined cerebellar alterations in a neonatal rat model of hypoxic-ischemic brain injury with or without hypoxic preconditioning (Pc). Between postnatal days 7 and 15, the cerebellum is still undergoing intense cellular proliferation, differentiation and migration, dendritogenesis and synaptogenesis. The expression of glutamate decarboxylase 1 (GAD67) and the differentiation factor NeuroD1 were examined as markers of Purkinje and granule cells, respectively. We applied quantitative immunohistochemistry to sagittal cerebellar slices, and Western blot analysis of whole cerebella obtained from control (C) rats and rats submitted to Pc, hypoxia-ischemia (L) and a combination of both treatments (PcL). We found that either hypoxia-ischemia or Pc perturbed the granule cells in the posterior lobes, affecting their migration and final placement in the internal granular layer. These effects were partially attenuated when the Pc was delivered prior to the hypoxia-ischemia. Interestingly, whole nuclear NeuroD1 levels in Pc animals were comparable to those in the C rats. However, a subset of Purkinje cells that were severely affected by the hypoxic-ischemic insult—showing signs of neuronal distress at the levels of the nucleus, cytoplasm and dendritic arborization—were not protected by Pc. A monoclonal antibody specific for GAD67 revealed a three-band pattern in cytoplasmic extracts from whole P15 cerebella. A ∼110 kDa band, interpreted as a potential homodimer of a truncated form of GAD67, was reduced in Pc and L groups while its levels were close to the control animals in PcL rats. Additionally we demonstrated differential glial responses depending on the treatment, including astrogliosis in hypoxiated cerebella and a selective effect of hypoxia-ischemia on the vimentin-immunolabeled intermediate filaments of the Bergmann glia. Thus, while both glutamatergic and GABAergic cerebellar neurons are compromised by the hypoxic-ischemic insult, the former are protected by a preconditioning hypoxia while the latter are not.

Disruptions to the cerebellar GABAergic system in juvenile guinea pigs following preterm birth

“…The decrease in GAD67+ Purkinje cells numbers identified in lobule X in both the preterm males and females coincided with a reduction in the internal granule cell layer width in both sexes. The internal granule cell layer comprises the glutamatergic neurons of the cerebellum (Benitez et al, 2014). These granule cells excite Purkinje cells of the cerebellum, which results in an increase in GABAergic inhibitory transmission (Cerminara et al, 2015).…”

Differential response of pineal microglia to surgical versus pharmacological stimuli

“…The mouse monoclonal anti‐neuronal class III β–tubulin (Tuj1; BioLegend, code #801201 and 801,202, RRID: http://scicrunch.org/resolver/AB_10063408; Covance, code #MMS‐435P, RRID: http://scicrunch.org/resolver/AB_2313773) is a well‐characterized antibody that was generated using microtubules from rat brain (see manufacturer's technical information). This antibody recognizes a single ∼50 kDa protein band on WBs (Benitez, Castro, Patterson, Muñoz, & Seltzer, ), and intact and degenerated nerve fibers by IHC (Hegarty, Hermes, Yang, & Aicher, ). We used this anti‐Tuj1 antibody to stain nerve fibers in the postnatal pineal gland (Ibañez Rodriguez et al, ; Yu et al, ).…”

Intrauterine Growth Restriction Alters the Postnatal Development of the Rat Cerebellum

“…This is the first study to demonstrate altered Bergmann glial fiber organization and density in IUGR pups. Hypoxia and undernutrition, both of which occur during IUGR have also been shown to affect Bergmann glia development [40,41] , suggesting that these factors may mediate the effect of IUGR on Bergmann glial fibers. Similarly, babies that are born preterm also have decreased Bergmann glial fiber density and fibers that do not extend to the pial surface of the cerebellum [42] .…”