Reactive oxygen species modulate the differentiation of neurons in clonal cortical cultures

Abstract

Reactive oxygen species (ROS) are important regulators of intracellular signaling. We examined the expression of ROS during rat brain development and explored their role in differentiation using cortical cultures. High levels of ROS were found in newborn neurons. Neurons produced ROS, not connected with cell death, throughout embryogenesis and postnatal stages. By P20, ROS-producing cells were found only in neurogenic regions. Cells with low levels of ROS, isolated from E15 brains by FACS, differentiated into neurons, oligodendrocytes, and astrocytes in clonal cultures. Neurons produced high ROS early in culture and later differentiated into two types: large pyramidal-like neurons that fired no or only a single action potential and smaller neurons that expressed nuclear calretinin and fired repeated action potentials. Antioxidant treatment did not alter neuron number but increased the ratio of small to large neurons. These findings suggest that modulation of ROS levels influences multiple aspects of neuronal differentiation.

Figure 1. Developmental expression of ROS and staining for cell death markers

Postnatal day 3 rat brain slices were stained for ROS production with CM-H₂DCF-DA (green, A–C), and counterstaining with either propidium iodide (red, B) or annexin V (red, C). Nuclei are stained with DAPI (blue, A, B). Yellow fluorescence results from the superposition of red and green and indicates when cells with high levels of ROS also express the cell death marker, propidium iodide (B). Annexin V stains the surface of cells (C, arrows) in the early to middle stages of apoptosis. Aggregates of annexin V were also observed within the slices (C). Slices from rat brains at the indicated ages and regions were stained for ROS production with CM-H₂DCF-DA (green). E, embryonic day; P, postnatal day; CX, cortex; OB, olfactory bulb; HC, hippocampus.

Figure 2. Physiology of cells with high levels of ROS

Patch clamp recordings were performed on high ROS-producing cells as described in Experimental Methods. Cells stained for ROS (B) were identified morphologically (A) and patched. (C) Firing pattern after current injection. (D) Firing rate as a function of current levels (F/I curve).

Figure 3. Characterization of clonal neurosphere cultures

(A–D) Single neurospheres were transferred to glass slides coated with poly-L-lysine and laminin and allowed to differentiate. Live cells were stained for the presence of ROS (green) at the indicated days after initiation of the differentiation process and nuclei were visualized with Hoescht dye (blue). At 0 days (A) and two days (B), low levels of ROS were present in cells that were also positive for the death marker propidium iodide (PI) (red). No live, healthy cells in the cultures showed signs of ROS production, indicated by the combined staining for ROS and PI, which appears as yellow staining. However, after four days (C), several PI negative cells stain for CM-DCF. By seven days (D), the number of ROS positive cells increased and the cells with the high ROS production started to acquire neuronal-like morphology. (E–H) After seven days of differentiation, cells were fixed and immunostained with specific antibodies against β III tubulin (E) (blue), O4 (F) (green) and GFAP (G) (red) (all three shown merged in H). Nuclei were stained with DAPI (E–G) (blue, but shown in yellow pseudocolor in panel E). All clonal cultures examined were multipotent. The majority of the cells were GFAP positive astrocytes (See Table 1). The number of neurons and oligodendrocytes was similar. No other cell types were present in these cultures. Scale bar=10 μ m.

Figure 4. Two morphologically distinct types of neurons are present in clonal and primary cortical cultures

(A–D) Clonal neurospheres were grown under differentiation conditions and immunostained for calretinin (green) and β III tubulin (red). Nuclei were counterstained with DAPI (blue). (E, F) Three-dimensional reconstructions obtained using confocal microscopy. Type I neurons (A, B, E, F) have large, flat somata with multiple processes and little expression of calretinin. Type II neurons (C, D, E, F) have small round somata and only 2–4 processes extend from them. One of the processes is long and thick while the others are thinner. (E, F) Confocal microscopy and three-dimensional reconstruction using IMARIS software show that calretinin is present in the nuclei and thick processes of type II neurons (E, F). The majority of calretinin immunostaining appears to reside in the nucleus (white staining in F represents the colocalization of the green and blue channels). Calretinin immunoreactivity was also detected in the nuclei of non-neuronal cells (F, arrowheads). Type I neurons have little calretinin staining in the perinuclear region and proximal processes but none in the nucleus (A, B). (G) In clonal cultures, type I neurons are the majority of the neuronal population, while type II neurons are fewer. In E15 primary cortical cultures grown for 4 days, the same morphologically distinct types of neurons were present in similar proportion to that seen for the clonal cultures. Scale bar=20μ m

Figure 5. Staining of primary E15 cortical cultures

A–C Cells were stained for β III-tubulin (red), calretinin (green), and DAPI (blue) and photographed at low magnification. These micrographs were used to count the numbers of neurons and neuronal types in primary cultures as quantified in Fig. 4 G. D, E. Cells were stained with the neuronal polarity markers MAP2 (red) and Tau (green) and imaged by confocal microscopy.

Figure 6. Electrophysiological characterization of type I and type II neurons in primary cultures

Recording made from E15 cortical neurons grown on glass coverslips for 14–20 days. (A) Cells were chosen for recording according to their morphological characteristics. As seen in DIC images, type I neurons had flat somata and several processes and type II neurons had round somata and a smaller number of processes. Bar = 20 μ m (B) Most type I neurons fired a single action potential (55% of cases) or none (27% of cases) (top panel), but the membrane potential showed some evidence of active currents. In contrast, type II neurons were able to fire multiple action potentials in response to current injection (81% of cases) (bottom panel).

Figure 7. ROS perturbation alters the numbers of type I and type II neurons

(A, C) Clonal cultures differentiated under control conditions contained fewer calretinin positive, type II neurons than calretinin negative, type I neurons. (B, D) Upon treatment with the antioxidant SOD-PEG (50 U/ml), there was a highly significant (p<0.001), two-fold increase in the number of type II neurons with a concomitant decrease in type I neurons (E). Calretinin staining increased in the processes of type I neurons in the presence of SOD-PEG, but was never found in the nucleus in these cells (D, arrows).

Figure 8. Schematic summarizing the major findings of the present work

A population of cells containing low levels of ROS from E14–15 rat cortex were isolated and shown by clonal cell culture to be multipotent neural progenitor cells. The high ROS-containing population was composed of neurons (Tsatmali et al., 2005). The multipotent progenitor cells gave rise to two types of neurons, the numbers of which were altered after antioxidant treatment.