We next developed methods to measure the intracellular activity of hippocampal neurons during behavior along the virtual linear track. Because the mouse’s head is stationary in the virtual reality setup, we were able to perform whole cell patch clamp recordings using a patch electrode with a long taper mounted on a standard micromanipulator positioned outside the mouse’s field of view (see Methods). We obtained recordings in awake mice17,18,30,31
as they ran on the spherical treadmill. Recordings lasted many minutes on average (7.7 ± 3.8 minutes; maximum, 20.4 minutes; n = 46 cells from 15 mice), during which time mice ran long distances at high speeds in the virtual environment (27 ± 5 m per minute; total distance, range: 125–458 m). We did not detect any major motion-induced artifacts in the electrophysiology recordings. Recordings could be performed from the same animal across multiple days (≥ 6 days per mouse; ≥3 days per hemisphere).
A subset of our whole cell recordings was made from place cells (overall firing rate = 2.2 ± 0.4 Hz; in-field firing rate = 7.3 ± 1.4 Hz; out-of-field firing rate = 1.5 ± 0.4 Hz; field size = 43 ± 13 cm; n = 8 cells from 8 mice; , Supplementary Figs. 2A, 3
). Approximately 36% of spontaneously active putative pyramidal neurons had a place field along the virtual track, which is consistent with estimates from extracellular recordings and immediate early gene studies in rats exploring real environments32,33
(see Methods). Place cells recorded intracellularly fired in theta bursts (high frequency bursts at >50 Hz occurring at theta frequencies of ~6–10 Hz), resulting in ISI distributions with peaks at <10 ms and ~130 ms, consistent with our extracellular measurements (c.f. Supplementary Fig. 4 and Fig. 3B
). The individual action potentials during a theta burst often occurred during the ascending phase of an underlying theta oscillation (). Spike amplitudes decreased within a burst without a change in the peak potential of the spike, suggesting that depolarizing intracellular oscillations contribute to intra-burst decreases in spike amplitude34
. Whole cell recordings from place cells therefore can be obtained in head-restrained mice behaving in virtual environments.
Ramp-like membrane potential depolarization inside place fields
Membrane potential theta oscillations in place cells
From our whole cell recordings of place cell activity, we examined two types of membrane potential dynamics proposed by the theoretical models of place cell function: ramps of depolarization and modulation of theta oscillations (). We first analyzed changes in the average baseline membrane potential during behavioral epochs inside and outside the place field. As a mouse approached the recorded cell’s place field, the average membrane potential, excluding action potentials, increased in a ramp-like manner and remained elevated until the place field was passed (). The ramp of depolarization often began before action potential firing in the place field commenced and in some cases reached a steady depolarization as large as ~10 mV (peak depolarization = 5.7 ± 2.9 mV; , Supplementary Figs. 5, 6
). These depolarization events occurred preferentially in place fields; the average membrane potential excluding action potentials was higher in place fields than outside of place fields (Vin-field
= 2.5 ± 0.5 mV, P < 0.0001; , Supplementary Fig. 2B
). Consistently, the baseline membrane potential and firing rate were strongly correlated (C = 0.55 ± 0.10, P < 0.001). On complete runs through the place field, ramps of depolarization were asymmetric with the peak depolarization shifted toward the end of the field (position of the peak depolarization = 72 ± 24% of the distance through the field, P < 0.05 vs. 50%; slope before peak = 3.0 mV per place field length, slope after peak = 5.4 mV per field length, P < 0.001; ). In contrast, firing rates were symmetric within the place field (position of the peak firing rate = 52 ± 17% of the distance through the field, P > 0.6 vs. 50%; slope before peak = 13.3 Hz per place field length, slope after peak = 13.6 Hz per field length, P > 0.4; ), perhaps because firing rates were highest on the ascending part of a ramp depolarization13
(Supplementary Fig. 7A
). An asymmetric ramp-like depolarization of the baseline membrane potential therefore is a subthreshold signature of place fields.
We next examined the modulation of the amplitude and phase of membrane potential oscillations occurring at theta frequencies during runs along the virtual track. We measured intracellular theta oscillations by band-pass filtering our membrane potential recordings from 6–10 Hz, after action potentials were removed (; Methods). When the mouse entered the recorded cell’s place field, the amplitude of intracellular theta oscillations increased (). Theta-band power in the membrane potential trace was higher in place fields than outside of place fields (Powerin-field
= 1.7 ± 0.4 mV2
= 0.8 ± 0.2 mV2
, P < 0.01; , Supplementary Figs. 2C, 8
). Consistently, theta oscillation amplitude and firing rate were highly correlated (C = 0.61 ± 0.09, P < 0.001). In contrast, the amplitude of membrane potential theta oscillations was similar at all spatial locations for putative CA1 pyramidal neurons that did not have a place field (Supplementary Fig. 9
) and for the LFP theta rhythm (Supplementary Fig. 10
To examine the modulation of the phase of intracellular theta, we compared intracellular theta fluctuations with LFP theta oscillations. We began by looking for phase precession of spike times relative to membrane potential theta oscillations. The intracellular phases of spike times did not change during runs through the place field, and the phase and position of spikes were not correlated (ΔPhase = 0.6 ± 10.4 degrees between the first and last eighth of the field, P > 0.9; C = −0.01 ± 0.08 between phase and position, P > 0.6; ). Because spike times advanced relative to LFP theta oscillations but not intracellular theta (c.f. , ), it is predicted that a phase shift between LFP and intracellular theta occurs during place field traversals. We therefore performed simultaneous whole cell and LFP recordings to directly compare the phases of intracellular and extracellularly-recorded theta. During runs through the place field, the phase difference between intracellular and LFP theta shifted such that the intracellular theta oscillation phase precessed relative to the LFP theta rhythm (C = −0.26 ± 0.12 between LFP phase and position for the times of intracellular theta peaks; n = 2 cells from 2 mice; , Supplementary Fig. 11A
). Consistently, the frequency of intracellular theta oscillations in the place field was higher than the frequency of LFP theta fluctuations (measured as a ratio of periods of intracellular theta to periods of LFP theta; Ratioin-field
= 0.97 ± 0.21, P < 0.01 vs. 1; Δfrequency = 0.23 Hz given a mean LFP frequency of 7.4 Hz; see Methods, Supplementary Fig. 11B
). In contrast, the frequencies of intracellular theta and LFP theta during epochs outside the place field were similar (Ratioout-of-field
= 1.01 ± 0.22, P > 0.2 vs. 1; Supplementary Fig. 11B
). Intracellular theta oscillations in place cells therefore were not constant in amplitude or phase (relative to LFP theta) throughout runs on the linear track; rather, membrane potential theta oscillations were dynamically modulated across positions in virtual space.
Ramp-like depolarizations of the membrane potential and increases in intracellular theta oscillation amplitude were present simultaneously, which can be demonstrated directly by filtering the membrane potential trace from DC-10 Hz (). Consistently, intracellular theta power and the baseline membrane potential were highly correlated (C = 0.52 ± 0.07, P < 0.001; , , Supplementary Fig. 2B–C
). To determine if ramps of depolarization trigger increases in theta amplitude, we injected ramps of current at the soma while the animal was running along the virtual track and measured changes in theta power. Theta power increased weakly with higher levels of depolarization; however, the increase in power was smaller than during runs through the place field (P < 0.01 at similar ΔV values; c.f. , and Supplementary Fig. 7B
). Ramp-like depolarizations of the membrane potential therefore were not sufficient to cause the increases in theta oscillation amplitude observed in place fields.
Our whole cell recordings revealed two additional subthreshold phenomena. First, in a fraction of our recordings we observed spikelets, brief small amplitude deflections of the membrane potential (2/8 place cells; amplitude = 7.4 ± 1.3 mV, full-width at half maximum = 1.6 ± 0.4 ms; 0.06 ± 0.05 spikelets per second; Supplementary Fig. 12A
. Second, bursts of action potentials were occasionally followed by large (~10–25 mV) depolarizations lasting up to 50–100 ms23
(Supplementary Fig. 12B
). These events sometimes contained broadened spikes of reduced amplitude, consistent with Ca2+
spikes recorded in slices36
. Further analysis is required to assess the prevalence, significance, and mechanisms of these events.