Behavioral and neurochemical consequences of cortical oxidative stress on parvalbumin-interneuron maturation in rodent models of schizophrenia

Abstract

Oxidative stress, in response to the activation of the superoxide-producing enzyme Nox2, has been implicated in the schizophrenia-like behavioral dysfunction that develops in animals that were subject to either neonatal NMDA receptor-antagonist treatment or social isolation. In both of these animal models of schizophrenia, an environmental insult occurring during the period of active maturation of the fast-spiking parvalbumin-positive (PV+) interneuronal circuit leads to a diminished expression of parvalbumin in GABA-inhibitory neurons when animals reach adulthood. The loss of PV+ interneurons in animal models had been tentatively attributed to the death of these neurons. However, present results show that for the perinatal NMDA-R antagonist model these interneurons are still alive when animals are 5-6 weeks of age even though they have lost their phenotype and no longer express parvalbumin. Alterations in parvalbumin expression and sensory-evoked gamma-oscillatory activity, regulated by PV+ interneurons, are consistently observed in schizophrenia. We propose that cortical networks consisting of faulty PV+ interneurons interacting with pyramidal neurons may be responsible for the aberrant oscillatory activity observed in schizophrenia. Thus, oxidative stress during the maturation window for PV+ interneurons by alteration of normal brain development, leads to the emergence of schizophrenia-like behavioral dysfunctions when subjects reach early adulthood.

Figure 1. Perinatal exposure to ketamine reduces the number of PV-expressing interneurons in the adult frontal cortex

Litters from two C57BL/6 females per treatment condition were injected subcutaneously with either saline or ketamine (30 mg/kg) on postnatal days 7, 9, and 11. Males, 5 for each treatment condition, were perfused with 4 % paraformaldehyde when they had reached 8 weeks of age, and their brains sliced for floating section immunohistochemistry as described (Behrens et al., 2007). Six consecutive slices encompassing the frontal region were immunostained with Vectastain for detection of parvalbumin using a polyclonal antibody (Swant, Switzerland). Cells in each region were counted as described (Dugan et al., 2009). All cells in the region were counted and total numbers per slice were corrected using Abercrombie’s correction algorithm (Abercrombie, 1946) and normalized to the saline samples. Bar graphs represent means ± SEM. *,#,& indicates statistical significance with respect to saline on each brain region as assessed by ANOVA followed by Tukey’s test (* F_(1,8) = 105.351, p < 0.05; ^# F_(1,8) = 25.460, p < 0.05; ^& F_(1,8) = 9.366, p = 0.05

Figure 2. Loss of PV-expression without death of the interneurons

The heterozygous progeny of G42 X C57BL/6 crossings was treated with saline or ketamine during the second postnatal week as described in figure 1 and the animals were perfused between the 5^th and 6^th week of age. The G42 line expresses GFP in approximately 50% of PV+ interneurons (Chattopadhyaya et al., 2004; Di Cristo et al., 2004). (A) Colocalization of GFP and PV was determined by confocal microscopy using a Zeiss Pascal system and AlexaFluor 568 as secondary antibody for detection of PV. (B) The prelimbic region on each slice was imaged across six consecutive slices, and across 16 mm on the Z axis. Images were then collapsed and the number of cells expressing PV or GFP were counted as described in figure 1 and normalized by the mean of the saline controls. As described in figure 1, there was a statistically significant decrease in the number of PV-expressing neurons, but there was no decrease in the GFP-expressing population. Since all GFP expressing neurons were also positive for PV in saline controls, as previously described (Chattopadhyaya et al., 2004; Di Cristo et al., 2004), these results suggest that the PV-interneuronal population is still present in these animals, but the neurons do not express their characteristic marker, parvalbumin. Bar graphs represent means ± SEM. * indicates statistical significance (p < 0.005) with respect to saline as determined by ANOVA (F_(3,12) = 12.278, p < 0.001) followed by Tukey’s pos hoc test.