Cholinergic modulation of the resonance properties of stellate cells in layer II of medial entorhinal cortex

Abstract

In vitro whole cell patch-clamp recordings of stellate cells in layer II of medial entorhinal cortex show a subthreshold membrane potential resonance in response to a sinusoidal current injection of varying frequency. Physiological recordings from awake behaving animals show that neurons in layer II medial entorhinal cortex, termed "grid cells," fire in a spatially selective manner such that each cell's multiple firing fields form a hexagonal grid. Both the spatial periodicity of the grid fields and the resonance frequency change systematically in neurons along the dorsal to ventral axis of medial entorhinal cortex. Previous work has also shown that grid field spacing and acetylcholine levels change as a function of the novelty to a particular environment. Using in vitro whole cell patch-clamp recordings, our study shows that both resonance frequency and resonance strength vary as a function of cholinergic modulation. Furthermore, our data suggest that these changes in resonance properties are mediated through modulation of h-current and m-current.

Fig. 1.

Application of carbachol significantly reduces resonance frequency and resonance strength of medial entorhinal cortex (mEC) stellate cells (SCs). A: representative examples of standard impedance amplitude profile (ZAP) response of stellate cells in control (black) and in 10 μM carbachol (gray). In both conditions, cells were held at −66 mV and injected with a 20 s ZAP stimulus with 65 pA amplitude, shown on the bottom of A. B: SC population averages of resonance frequency (Hz) and resonance strength in control and carbachol conditions. C: average impedance profile in control (black) and carbachol (gray) across all cells.

Fig. 2.

Control experiments show that the change in resonance properties result from activation of muscarinic acetylcholine receptors and are not caused by whole cell patch-clamp–induced washout and channel rundown. A and B: examples of the voltage response of SCs generated from injection of a ZAP stimulus of 65 pA amplitude. A: the voltage response was measured in 1 μM atropine (black) and after 10 min bath application of 10 μM carbachol with 1 μM atropine (gray). B: the voltage response was measured in control (black) and in time control condition (gray), which consisted of waiting 15 min after the last control ZAP stimulus was injected. C: the frequency dependent impedance profile was averaged across all cells in atropine (black) and in atropine with carbachol (gray). D: the impedance profile was measured in control condition (black) and in time control condition (gray). E: average change in resonance frequency (Hz) and resonance strength across all atropine with carbachol (Atro + CCh) control experiments is contrasted with the change in resonance frequency after application of carbachol (CCh), as presented in Fig. 1. F: average change in resonance frequency (Hz) and resonance strength across all time control experiments is contrasted with the change in resonance frequency after application of carbachol (CCh; from Fig. 1).

Fig. 3.

Stellate cells across the dorsal to ventral axis of mEC show differential changes in resonance frequency as a function of mAChR activation. A: SCs in ventral mEC show a larger percentage decrease in resonance frequency on application of carbachol. B: resonance frequency is lower in control condition SCs in ventral mEC (5.64 ± 0.62 Hz, n = 8) compared with SCs in dorsal mEC (7.28 ± 0.35 Hz, n = 15; P < 0.05). Resonance frequency measured in carbachol is lower in SCs in ventral mEC (3.68 ± 0.45 Hz, n = 8) compared with SCs in dorsal mEC (5.71 ± 0.28 Hz, n = 15; P < 0.01). The decreases shown in resonance frequency in ventral SCs from the control to the carbachol condition and in dorsal SCs from the control to the carbachol condition are both statistically significant (P < 0.01).

Fig. 4.

Simulation results from a biophysical conductance–based model predicts the channel physiology that could mediate the muscarinic acetylcholine receptor (mAChR)-dependent changes in resonance frequency and resonance strength. A: resonance properties were measured as a function of the maximum conductance values of the h-current (diamond), m-current (square), and leak current (triangle). At a holding potential of −58 mV, the resonance frequency is shown on the left and resonance strength is shown on the right. B: individual simulations of ZAP protocols are shown at −58 mV in control, h-current block, m-current block, and leak-current block from top to bottom. C: at a holding potential of −68 mV, the resonance frequency is shown on the left and resonance strength is shown on the right, with the same colors for manipulations of currents in A. D: individual simulations of ZAP protocols are shown at −68 mV in control, h-current block, m-current block, and leak-current block from top to bottom. E: resonance frequency (left) and resonance strength (right) were measured as a function of the h-current time constant. The h-current time constant was scaled by factors of 0.5, 1, and 2.

Fig. 5.

Analysis of membrane potential sag response suggests mAChR-dependent modulation of h-current. A: to measure the membrane potential sag response, SCs were hyperpolarized from −65 mV by injecting −200 to −300 pA current for 3 s. Responses were measured in control conditions (left) and after 10 min bath application of 10 μM carbachol solution (right). The sag amplitude is measured as the difference between the initial hyperpolarized membrane potential and the steady-state membrane potential. The response was fit with a dual exponential to measure time course of sag response. B: the averages across all SCs show an increase in the time constant (left) and a significant decrease in sag amplitude (right) from the control to the carbachol condition.

Fig. 6.

Pharmacological blockade of cyclic nucleotide-regulated (HCN) channels shows that cholinergic modulation of the resonance properties and the sag response is dependent on the h-current. A: ZAP protocols were run in control (left) and in the presence of 10 μM ZD7288 (right). B: resonance frequency was reduced from 6.05 ± 0.66 Hz in control (black) to 1.52 ± 0.13 Hz in the ZD7288 (gray) (P < 0.01, n = 6). Resonance strength was reduced from 1.54 ± 0.07 in control (black) to 1.19 ± 0.06 in ZD7288 (gray) (P < 0.01, n = 6). Addition of carbachol to ZD7288 (red) gave a resonance frequency 1.52 ± 0.12 Hz and a resonance strength of 1.14 ± 0.04. C: the voltage response caused by hyperpolarizing current steps is shown in 10 μM ZD7288 (black) and in 10 μM ZD7288 with 10 μM carbachol (gray). D: blockade of H-channels reduced the sag amplitude to 0.084 ± 0.034 mV (black, solid), and subsequent application of carbachol had no significant effect on the sag amplitude (0.088 ± 0.12 mV; gray solid). The change in sag amplitude from control ACSF to carbachol was 1.29 ± 0.22 mV, and this was significantly different from the change from ZD 7288 to ZD7288 with carbachol (−0.004 ± 0.11 mV; P < 0.01, n = 6; D, right).

Fig. 7.

Pharmacological manipulation of KCNQ channels shows a significant m-current–dependent resonance at depolarized membrane potentials. A: individual ZAP responses are shown at −58 mV in control (black) and after 10 min bath application of 10 μM XE991 and again at −68 mV for both conditions. B: the plot of the impedance profile depicts the response of the SCs in the control (black) and in the XE991 condition (gray) at the more depolarized membrane potentials of −58 mV (left) and at the more hyperpolarized membrane potentials of −68 mV (right). C: population averages show that the block of m-current decreases resonance frequency from control to XE991 condition at a membrane potential of −58 mV (P < 0.05). In contrast, resonance frequency at −68 mV membrane potentials does not show changes that are significantly different from 0. Resonance strength decreases at −58 mV and does not change significantly at −68 mV.