Fig 1.
Photostimulation of different parts of a cell expressing channelrhodopsin-2.
A: Confocal image of mouse cortical neurons expressing ChR2. Mice were transfected in utero using intraventricular electroporation with two plasmids, one containing red fluorescent protein that stains cytoplasm and is best visible in cell bodies, and another one containing ChR2 fused with Venus (green/yellow) that stains cell membrane, best visible in dendrites. B: Responses of a layer 2/3 pyramidal neuron expressing ChR2 to a step of depolarizing current injected through the recording electrode or induced by light pulse of a 488 nm laser. C: Light-induced responses in a ChR2 expressing neuron to 11 ms light pulses of different intensities illuminating soma region. On the left, responses to subthreshold and suprathreshold light stimuli are superimposed. D: Light-induced responses in the same cell to short (2 ms) illumination of distal dendrites at subthreshold intensity and at high light intensity that induced dendritic spike.
Fig 2.
Membrane potential responses to sine-wave modulation of light intensity express stronger attenuation of high frequencies than responses to intracellular current injection.
A: Membrane potential responses (blue traces) of a layer 2/3 pyramidal neuron expressing ChR2 to a 488 nm diode light modulated by sine-wave signals at 10, 50 and 100 Hz (light blue traces). The amplitude of the sine-wave modulation of light intensity was kept constant. B: Membrane potential responses (red traces) of a neuron to injection of sine-wave current at 10, 50 and 100 Hz (green traces). Two-electrode experiment. One electrode was used for current injection, and another electrode for recording of membrane potential responses. The amplitude of the sine-wave current was kept constant. C: Attenuation of the amplitude of membrane potential modulation in responses to sine-wave signals of different frequencies injected through the intracellular electrode or induced by photostimulation.
Fig 3.
Frequency response of the membrane potential to fluctuating current injected through intracellular electrode or photoinduced in somatic region and in distal dendrites.
A: Membrane potential responses of layer 2/3 pyramidal neuron to injection of subthreshold fluctuating current through intracellular electrode. Two-electrode experiment. B: Membrane potential responses of the same neuron to illumination of somatic region or distal dendrites by the light from 488 nm diode with intensity modulated by the same fluctuating current as used for intracellular injection. The illumination field for dendritic stimulation was shifted ~150 μm from the soma. For dendritic stimulation light intensity was increased 2.5 times in order to induce membrane potential fluctuations of the the same amplitude range. C: Normalized power spectra of the current used for injection or modulation of light intensity, and of membrane potential responses to intracellular current injection and to photostimulation of the somatic region and of distal dendrites.
Fig 4.
Frequency transfer functions calculated from responses of a neuron to fluctuating current, either injected through intracellular electrode or photoinduced in somatic region and in distal dendrites.
A: Membrane potential and spike responses to intracellular injection of fluctuating current. DC current was added to maintain target firing rate of ~5 Hz. B: Membrane potential responses to illumination of the somatic region or distal dendrites by light modulated by the same fluctuating current as used for intracellular injection. DC current was injected through the somatic electrode to maintain target firing rate. C: Frequency transfer functions calculated from responses to fluctuating current, injected intracellularly or photoinduced in the soma or in distal dendrites. Transfer functions were calculated using modified Higgs & Spain [6] method. Faint lines show 95th percentile of N = 500 transfer functions calculated using shuffled spike timings. Transfer factors are considered significant when above the 95th percentile of shuffled-spike values; transfer function is cut at the intersection with the 95th percentile curve [7].
Fig 5.
Responses of a layer 2/3 pyramidal neuron to proximal and distal photostimulation, and electrical stimulation of proximal or distal synaptic inputs.
A: Membrane potential response to fluctuating current injected through the intracellular recording electrode. B, C: Membrane potential response to a mixture of intracellular injection of the same fluctuating current and photoinduced optical EPSCs (oEPSCs) produced by 2 ms pulses of 488 nm light illuminating somatic region (B) or distal dendrites (C). Dashed vertical line shows the timing of an oEPSC which led to generation of an additional action potential. Note that other oEPSCs did not lead to additional spikes. D: Response of pyramidal neuron to intracellular injection of fluctuating noise current with immersed small artificial aEPSCs. E: A scheme of optical and electrical stimulation of a layer 2/3 pyramidal neuron. Black line on top shows cortical surface. Electrical stimuli were applied through patch stimulation electrodes located in layer 1 or in layer 2/3. For optical stimulation, either somatic region or distal part of apical dendrite was illuminated, as indicated by the circles. F: EPSCs evoked by electrical stimulation and photoinduced oEPSCs. All responses were recorded by intracellular pipette in the soma. Note that EPSCs induced by electrical or optical stimulation in layer 1 have slower onsets, indicative of their more distal origin, than EPSCs induced by electrical or optical stimulation in layer 2/3. G: PSTHs of spike responses to EPSCs immersed in fluctuating current. EPSCs were photoinduced in the somatic region or in distal dendrites, or produced by intracellular current injection. Note that in all three cases, the increase of the firing rate follows the dynamics of EPSC onset. Abscissa is population firing rate in Hz.