Reduction of cortical parvalbumin-expressing GABAergic interneurons in a rodent hyperoxia model of preterm birth brain injury with deficits in social behavior and cognition

Abstract

The inhibitory GABAergic system in the brain is involved in the etiology of various psychiatric problems, including autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD), and others. These disorders are influenced not only by genetic but also by environmental factors, such as preterm birth, although the mechanisms underlying are not known. In a translational hyperoxia model, exposing mice pups at age P5 to 80% oxygen for 48 hours to mimic a steep rise of oxygen exposure caused by preterm birth from in utero into room air, we documented a persistent reduction of cortical mature parvalbumin expressing interneurons until adulthood. Developmental delay of cortical myelin was observed together with decreased expression of oligodendroglial glial cell-derived neurotrophic factor (GDNF), a factor being involved in interneuronal development. Electrophysiological and morphological properties of remaining interneurons were unaffected. Behavioral deficits were observed for social interaction, learning, and attention. These results elucidate that neonatal oxidative stress can lead to decreased interneuron density and to psychiatric symptoms. The obtained cortical myelin deficit and decreased oligodendroglial GDNF expression indicate an impaired oligodendroglial-interneuronal interplay contributes to interneuronal damage.

Neonatal Oxidative Stress Impairs Cortical Synapse Formation and GABA Homeostasis in Parvalbumin-Expressing Interneurons

“…As previously described [ 30 ], total cortical RNA was isolated by acidic phenol/chloroform extraction (peqGold RNAPure, PEQLAB Biotechnologie, Erlangen, Germany) from mouse cortical samples. After DNase (Qiagen, Hilden, Germany) pretreatment, two μ g of total RNA was reverse transcribed (M-MLV Reverse Transcriptase, Promega, Walldorf, Germany) for cDNA synthesis.…”

“…A fixed amount of 20 μ g of isolated protein from mouse cortex was separated by SDS-PAGE using a 4–20% Criterion™ TGX™ Precast Mini/Midi Protein Gel (Bio-Rad, Feldkirchen, Germany). As described elsewhere [ 30 ], blotting (Trans-Blot Turbo Transfer System, Bio-Rad) was performed to a nitrocellulose membrane. Unspecific binding sites were blocked (Roti-Block, Car-Roth, Karlsruhe, Germany), and membranes were thereafter incubated with primary antibodies as follows: anti-nitrotyrosine (Millipore, 06-284, 1 : 1000), anti-GAD1/GAD67 (Novus Biologicals, NBP102161, 1 : 1000), anti-phosphoinositide 3-kinase (Upstate, 06-497, 1 : 1000), anti-phospho-AKT (Cell Signaling Technologies, 9271, 1 : 500), and anti- β -AKTIN (Sigma, A5316, 1 : 5000), followed by appropriate secondary antibodies conjugated with horseradish peroxidase (donkey anti-rabbit Pierce #31458, 1 : 5000; rabbit anti-mouse Dako P0260, 1 : 5000; goat anti-chicken Invitrogen A16054, 1 : 5000) and detection by chemiluminescence (PerkinElmer, USA).…”

“…Unspecific binding sites were blocked (Roti-Block, Car-Roth, Karlsruhe, Germany), and membranes were thereafter incubated with primary antibodies as follows: anti-nitrotyrosine (Millipore, 06-284, 1 : 1000), anti-GAD1/GAD67 (Novus Biologicals, NBP102161, 1 : 1000), anti-phosphoinositide 3-kinase (Upstate, 06-497, 1 : 1000), anti-phospho-AKT (Cell Signaling Technologies, 9271, 1 : 500), and anti- β -AKTIN (Sigma, A5316, 1 : 5000), followed by appropriate secondary antibodies conjugated with horseradish peroxidase (donkey anti-rabbit Pierce #31458, 1 : 5000; rabbit anti-mouse Dako P0260, 1 : 5000; goat anti-chicken Invitrogen A16054, 1 : 5000) and detection by chemiluminescence (PerkinElmer, USA). Loading control β -actin was used as an internal reference [ 30 ].…”

“…Anesthetized mice were transcardially perfused first with phosphate-buffered saline (PBS) and then again with 4% paraformaldehyde (PFA) solution. After dissection, brains were furthermore fixed in 4% PFA at 4°C overnight as previously described [ 30 ]. Washing was performed using PBS, followed by cryoprotection in sucrose solutions at 4°C, starting with 5% sucrose dissolved in PBS for one hour, then 10% sucrose solution overnight, and then with 30% sucrose solution until the organs dropped to the bottom of the glass.…”

“…In our study, we are using a neonatal oxidative stress model of preterm birth brain injury, in which five-day-old (P5) mouse pups are exposed to hyperoxia (80% oxygen) for 48 hours, causing a 3- to 4-fold increase of oxygen levels [ 13 , 29 ]. In a previous study, we could demonstrate a significant reduction of cortical PVALB+ interneurons, both acutely and during further developmental until adulthood, which was accompanied by altered behavior being caused by neonatal hyperoxia/oxidative stress exposure [ 30 ]. Here, we aim to define the impact of neonatal oxidative stress on GABA homeostasis and synapse formation as key aspects for functional maturation of cortical PVALB+ interneurons playing a role in psychiatric disorders often diagnosed in preterm infants.…”