Functional neurogenesis in the adult hippocampus

Abstract

There is extensive evidence indicating that new neurons are generated in the dentate gyrus of the adult mammalian hippocampus, a region of the brain that is important for learning and memory. However, it is not known whether these new neurons become functional, as the methods used to study adult neurogenesis are limited to fixed tissue. We use here a retroviral vector expressing green fluorescent protein that only labels dividing cells, and that can be visualized in live hippocampal slices. We report that newly generated cells in the adult mouse hippocampus have neuronal morphology and can display passive membrane properties, action potentials and functional synaptic inputs similar to those found in mature dentate granule cells. Our findings demonstrate that newly generated cells mature into functional neurons in the adult mammalian brain.

Figure 1

Dividing cells in the adult mouse dentate gyrus express early neural markers at 48 h after virus injection. a, Confocal micrograph (a merged image of 15 1-µm optical sections) of GFP expression in the dentate gyrus in a section that was labelled for the neuronal marker NeuN (red). No co-labelling of GFP⁺ cells with NeuN was observed. b, Single confocal plane of a cluster of GFP⁺ cells labelled with the early neuronal marker Tuj1-β (red). c, Single confocal plane of a GFP⁺ cell with immunoreactivity for the progenitor marker NG2 (red). In b and c, nuclei (DAPI) and GFAP are blue.

Figure 2

GFP⁺ cells in the dentate gyrus 4 weeks after virus injection express mature neuronal markers. a, Overview of GFP expression in the dentate gyrus in a section (a merged image of 27 1-µm optical sections) that was labelled for the neuronal marker NeuN (red). b, GFP⁺ cell (a merged image of 18 1-µm optical sections) immunoreactive for NeuN. c, Single confocal plane of the same cell, showing co-localization of GFP and NeuN.

Figure 3

Newly generated neurons receive synaptic inputs. a, Confocal photomicrograph (a merged image of 10 0.5-µm optical sections) of a GFP⁺ cell, immunolabelled for calbindin (blue) and synaptophysin (red). Adjacent panels (merged images of 4 0.5-µm optical sections) show co-localization with calbindin and synaptophysin. b–e, Visualization of spines in GFP⁺ cells (merged images of 12–33 optical sections of 0.5 µm). The boxed areas in the insets correspond to the enlarged images of the dendrites. Cells were analysed 4 months (b, c) or 4 weeks (d, e) after virus injection. f, Electron micrograph of synaptic terminals (arrows) on the soma of a GFP⁺ neuron (asterisk) in the granule cell layer.

Figure 4

Newly generated cells display neuronal electrophysiological properties. a, Fluorescent micrographs of a GFP⁺ cell filled with Alexa through a recording pipette. b, Membrane potential in response to depolarizing currents (I_m; 500 ms, 20–400 pA) recorded under current clamp at the resting potential (−73.5 mV). Numbers on the left indicate stimulus size. Scale: 25 mV, 50 ms. c, Action potential recorded at 80 pA. d, Firing rate versus current curve. e, Spontaneous postsynaptic currents recorded under voltage clamp (−80 mV). Scale: 20 pA, 100 ms. f, Confocal micrographs taken from a single plane (1 µm) after fixation of the slice. g, Overview of the dorsal blade of the granule cell layer (merged z-series of 23 planes).

Figure 5

Newly generated neurons are functionally similar to mature dentate granule cells. Consecutive recordings of a GFP⁺ neuron (right) and an adjacent granule cell (left) are shown. The GFP⁺ neuron (C_m = 32 pF) was smaller and, therefore, younger than the unlabelled neuron (C_m = 60 pF). a, Fluorescence micrographs of both cells filled with Alexa (left panels). The right panel is a confocal micrograph taken after fixation, with GFP (green), Alexa (red) and DAPI (blue) visualized. b, Membrane potential in response to depolarizing current steps. Stimulus size is indicated on the left. Scale: 50 mV, 50 ms. c, Postsynaptic currents evoked by paired-pulse stimulation (100 µA, 0.2 ms, with a 50-ms interval) of the perforant path. The position of the stimulating electrode was identical for both neurons. Transients before the traces are stimulus artifacts. Scale: 100 pA, 25 ms (left); 40 pA, 25 ms (right). d, Spiking rate versus current curve. Insets show a single action potential.