Electrophysiological Differentiation of New Neurons in the Olfactory Bulb

Newly generated cells in different layers of the olfactory bulb. a, A newborn eGFP+ PG cell (green) around a glomerulus at P109, 3 weeks after virus injection. Calretinin-labeled (red) and tyrosine hydroxylase-labeled (blue) PG cells outline the glomerulus. Scale bar, 20 μm. b, GFP+ granule cell in the mitral cell layer (red, Nissl staining; P52, 37 d survival). Scale bar, 40 μm. c, GFP+ cells in the granule cell layer (red, TH; magenta, TOPRO3; P52, 37 d survival). Scale bar, 40 μm.

Properties of newborn PG cells compared with those of older neurons of the same type. a, Families of whole-cell currents in response to voltage steps from -50 to 80 mV after 250 msec preconditioning at -120 mV (top) and -50 mV (bottom) (P28, 13 d after virus injection). b, Voltage responses to injected currents (from -40 to 70 pA in steps of 10 pA) from a potential of -70 mV. c, Membrane potential as a function of injected current; average potential during the last 100 msec of the depolarizing pulse. a-c, GFP+ PG cell. d, Families of whole-cell currents in control PG cells in response to the same protocols as in a. e, f, I-V and conductance-voltage relationships of sodium current (○, controls; •, GFP+ cells). Maximal values of g_Na were 11.13 nS in controls and 15.85 nS in GFP+ PG cells. The voltage dependencies of g_Na were described by Boltzmann equations with midpoints and slopes of -34.4 and 6.76 mV for controls and -37.5 and 6.48 mV for GFP+ PG cells. g, h, I-V and conductance-voltage relationships of A current (○, controls; •, GFP+ cells). Maximal values of g_A were 8.82 nS in controls and 10.5 nS in GFP+ PG cells. The voltage dependencies of g_A were described by Boltzmann equations with midpoints and slopes of -29.3 and 11.15 mV for controls and -27.4 and 15.1 mV for GFP+ PG cells.

Maturation of voltage-dependent currents in GFP+ cells. The recording from a cell in the positions is indicated by arrows. a, P32, 9 d survival. b, P28, 10 d survival. c, P31, 11 d survival.

Electrophysiological properties of a restricted group of GFP+ cells in the glomerular layer (see Results for explanation). a, Voltage-clamp recordings; depolarizing steps to potentials from -70 to 60 mV after 300 msec preconditioning to -120 mV. b, Current-clamp responses to the injection of depolarizing current pulses showing repetitive action potentials. c, Spontaneous activity at rest, leading to repetitive action potentials. d, Giant depolarizing synaptic potentials, occasionally seen in GFP+ cells. e, Spontaneous synaptic currents blocked by 1 mm kynurenate (recorded at P20, 10 d survival). f, Responses to focal application of GABA and glutamate (both 100 μm; P39, 17 d survival). Decay time constants were 1.64 sec for GABA and 2.33 sec for glutamate. g-i, Current-voltage relationships for I_Na, I_K(V), and I_A. I_Na was measured as peak inward current, I_K(V) is the asymptotic current evoked by depolarizing steps from a holding potential of -50 mV in the presence of 0.1 μm TTX, and I_A is the peak outward current obtained by subtracting the I_K(V) currents from total outward currents evoked by depolarizing steps at identical potentials from a holding potential of -120 mV. j-l, Conductance-voltage relationships obtained by dividing the I-V data shown in g--i by the corresponding driving forces. The g-V relationships could be fitted by Boltzmann equations having the following parameters (amplitude, midpoint, and slope): g_Na, 18.3 nS, -39.0 mV, 4.91 mV; g_K(V), 8.26 nS, -10.4 mV, 13.7 mV; g_A, 16.7 nS, -10.2 mV, 18.6 mV.

Electrophysiological properties of GFP+ cells in the granule cell layer. a, Current-clamp recordings; responses to the injection of squared current pulses ranging from -50 to 40 pA in steps of 10 pA. b, Voltage-clamp recordings; responses to depolarizing voltage steps ranging from -70 to 30 mV after 300 msec preconditioning at -120 mV. c, d, Current-voltage and conductance-voltage relationships for sodium current, measured at the peak after suppression of the outward current with 20 mm TEA. e, f, Current-voltage and conductance-voltage relationships for delayed-rectifier potassium current, measured at the end of 40 msec depolarizing steps.

Synaptic properties of newly generated PG cells. a, Spontaneous excitatory synaptic currents in a newborn cell in the glomerular layer (P27, 12 d survival). This activity was blocked by picrotoxin but not by kynurenate. b, Action potentials in response to stimulation of the ON (P26, 12 d survival). c, Synaptic currents evoked by stimulation of the ON (same cell as b). Note that the delays between the stimulus artifact and the responses shown in b and c suggest a monosynaptic contact between ON and PG cells. d, Responses to focal application of 100 μm GABA and 100 μm glutamate in GFP+ PG cells (P47, 27 d survival). e, Responses to focal GABA application (100 μm) recorded under voltage-clamp conditions in gramicidin perforated patches. The three recordings were obtained at -50, -70, and -90 mV, and their peak amplitudes are shown in f (P47, 27 d survival). f, Linear regression of the peak responses shown in e, indicating a reversal potential at -65.81 mV, corresponding to an intracellular chloride concentration of 9.75 mm. g, Example of dose-dependent GABA-induced currents in GFP+ PG cells (responses to 5, 10, 50, 100, and 200 μm). h, GABA dose-response curves for GFP+ and control PG cells (○, controls; •, GFP+ cells). EC₅₀ were 28 and 27.7 μm, and the Hill coefficients were 1.55 and 1.23 for controls and GFP+ cells, respectively.