Integration of new neurons into functional neural networks

Abstract

Although it is established that new granule cells can be born and can survive in the adult mammalian hippocampus, there remains some question concerning the functional integration of these neurons into behaviorally relevant neural networks. By using high-resolution confocal microscopy, we have applied a new strategy to address the question of functional integration of newborn neurons into networks that mediate spatial information processing and memory formation. Exploration-induced expression of the immediate-early gene Arc in hippocampal cells has been linked to cellular activity observed in electrophysiological recordings under the same behavioral conditions. We investigated whether mature (5-month-old), newborn granule cells express Arc in response to a discrete spatial experience by detecting the expression of Arc in combination with NeuN (neuron-specific nuclear protein)-positive and bromodeoxyuridine-positive cells. We found that mature new granule cells do indeed express Arc in response to an exploration experience, supporting the idea that these cells are well integrated into hippocampal circuits. The proportion of mature newborn neurons that expressed Arc in response to exploration, however, was significantly higher (approximately 2.8%) than the proportion of cells that expressed Arc in the already existing population of granule cells (approximately 1.6%; p < 0.01). This finding extends previous data suggesting that the cellular physiology of newborn granule neurons differs from that of the existing population by indicating that these properties are retained in mature adult-generated neurons. Thus, these data have interesting implications for network models of spatial information processing and the role of hippocampal circuits in memory, indicating that mature new neurons are selectively recruited into hippocampal cell assemblies in higher proportions than older cells.

Figure 1.

A, C, Whole DG reconstructions from a caged control (A) and an exploration-treated animal (C), assembled with 25× overlapping confocal images. Granule cells are shown labeled with NeuN in blue (Cy5), Arc in red (Cy3), and BrdU in green (FITC). Note that the DG shows sparse expression of Arc protein in response to spatial exploration, as reported previously (Chawla et al., 2005; Ramirez-Amaya et al., 2005); however, more activated cells are observed in the exploration animal compared with the caged control (C vs A). B, D, Confocal images (40×) from the DG showing spatial exploration-induced Arc expression in a new (BrdU+) granule neuron (NeuN+) 5 months of age. When BrdU and Arc are colocalized, the combination appears orange, whereas NeuN and Arc colocalization appears pink. The 40× images were taken from three-dimensional reconstruction image stacks obtained from software from the Zeiss LSM confocal microscope. To verify that BrdU does colocalize with Arc, every time a BrdU cell appeared to express Arc, a three-dimensional image was taken. Only those cells that exhibited cytoplasmic Arc staining surrounding the BrdU nuclei were considered to be Arc-expressing BrdU neurons. Scale bars: A, C, 100 μm; B, D, 10 μm.

Figure 2.

A, The proportion of granule neurons (NeuN+) in which BrdU was detected, from the total population of granule cells. Differences were found in the upper versus lower blades of the DG. B, The proportion of Arc-positive cells detected from the total population of granule cells. Significant differences were found in Arc expression between exploration and caged control groups only in the upper blade. C, The proportion of cells that show BrdU and Arc colocalization in the upper and lower blades of the DG. Note the low, but statistically reliable, proportion of cells that showed both Arc and BrdU labeling. D, The proportion of Arc-expressing cells in the population of newborn neurons is compared with the proportion of Arc-expressing cells in the population of already existing granule neurons. Significant differences were found in the proportion of Arc-expressing cells between newborn and already existing granule cells only in the exploration group. ANOVA, *p < 0.05 and **p < 0.01; t test, ^††p < 0.01. Error bars indicate SEM.