Differential modulation of repetitive firing and synchronous network activity in neocortical interneurons by inhibition of A-type K(+) channels and Ih

Abstract

GABAergic interneurons provide the main source of inhibition in the neocortex and are important in regulating neocortical network activity. In the presence 4-aminopyridine (4-AP), CNQX, and D-APV, large amplitude GABAA-receptor mediated depolarizing responses were observed in the neocortex. GABAergic networks are comprised of several types of interneurons, each with its own protein expression pattern, firing properties, and inhibitory role in network activity. Voltage-gated ion channels, especially A-type K(+) channels, differentially regulate passive membrane properties, action potential (AP) waveform, and repetitive firing properties in interneurons depending on their composition and localization. HCN channels are known modulators of pyramidal cell intrinsic excitability and excitatory network activity. Little information is available regarding how HCN channels functionally modulate excitability of individual interneurons and inhibitory networks. In this study, we examined the effect of 4-AP on intrinsic excitability of fast-spiking basket cells (FS-BCs) and Martinotti cells (MCs). 4-AP increased the duration of APs in both FS-BCs and MCs. The repetitive firing properties of MCs were differentially affected compared to FS-BCs. We also examined the effect of Ih inhibition on synchronous GABAergic depolarizations and synaptic integration of depolarizing IPSPs. ZD 7288 enhanced the amplitude and area of evoked GABAergic responses in both cell types. Similarly, the frequency and area of spontaneous GABAergic depolarizations in both FS-BCs and MCs were increased in presence of ZD 7288. Synaptic integration of IPSPs in MCs was significantly enhanced, but remained unaltered in FS-BCs. These results indicate that 4-AP differentially alters the firing properties of interneurons, suggesting MCs and FS-BCs may have unique roles in GABAergic network synchronization. Enhancement of GABAergic network synchronization by ZD 7288 suggests that HCN channels attenuate inhibitory network activity.

Keywords: 4-AP; A-type K+ channels; GABAergic interneurons; HCN channels; Ih; Martinotti cells; basket cells; neocortex; synchronization.

FIGURE 1

Identification of neocortical GABAergic interneurons in VGAT-Venus transgenic rats. (A) Confocal image of Venus-expressing interneurons in upper layers of rat neocortex. Morphologically distinct subtypes of interneurons can be identified. (B) Confocal image of Venus-expressing interneurons (yellow) and biocytin labeled interneurons labeled (red). Neurons were labeled with biocytin during whole cell recording. Distinct morphological differences were observed. Leftmost neuron had characteristics of a Martinotti cell (MC) whereas rightmost neuron was classified as a fast spiking basket cell (FS-BC). (C) Response of MC to depolarizing and hyperpolarizing current pulses demonstrate typical repetitive firing properties and prominent sag responses. (D) Representative response of FS-BC to depolarizing and hyperpolarizing current pulses. High frequency repetitive firing, prominent after hyperpolarizations and lack of a pronounced sag response were typical of fast spiking basket cells.

FIGURE 2

Effects of 4-AP on action potential (AP) properties of FS-BCs. (A) Representative firing from a L2/3 FS-BC during a 400 pA depolarizing current pulse before (black) and after (red) bath application of 100 uM 4-AP. 4-AP induced spike widening and decreases in fAHP amplitude. (B) Superimposition of APs from A before and after 4-AP demonstrates significantly increased AP duration and decreased AHP amplitude. (C) Summary plot showing that bath application of 4-AP significantly increases AP half width of FS-BCs (n = 10). (D) Summary plot illustrating that 4-AP significantly decreases AHP amplitudes in FS-BCs (n = 10). ^∗p < 0.05.

FIGURE 3

4-aminopyridine induced changes in the repetitive firing properties of FS-BCs. (A) Representative response of a FS-BC to a 400 pA depolarizing current pulses before (black) and after (red) bath application of 4-AP. (B) Expanded view of the initial firing at the onset of responses shown in A. 4-AP induced an increase in the initial firing frequency. (C) Summary plot illustrating that bath application of 4-AP significantly increases the initial firing frequency at the onset of a depolarizing current (n = 10). (D) Summary plot showing that the accommodation ratio, calculated by dividing the first interspike interval by the last interspike interval, was significantly increased after bath application of 4-AP, indicating that accommodation was increased (n = 10). ^∗p < 0.05.

FIGURE 4

Action potential (AP) properties of b-AC Martinotti cells (b-AC MCs) are modified in the presence of 4-AP. (A) Representative AP firing of a L2/3 b-MC during a 475 pA depolarizing current pulse before (black) and after (red) bath application of 4-AP. (B) Superimposition of APs from A before and after bath application of 4-AP showing spike widening and burst firing in presence of 4-AP. (C) Summary plot demonstrates that bath application of 4-AP significantly increases AP duration in MCs (n = 10). (D) Summary graph illustrating that the AHP after each AP in b-AC MCs was significantly reduced by bath application of 4-AP (n = 10). ^∗p < 0.05.

FIGURE 5

Alterations in repetitive firing of b-AC MCs following bath application of 4-AP. (A) Representative response of a b-AC MC to a 475 pA depolarizing current pulse before (black) and after (red) bath application of 4-AP. (B) An expanded view of the initial firing pattern at the onset of a 475 pA depolarizing current pulse before and after bath application of 4-AP. Enhanced, continuous, burst firing was seen in presence of 4-AP. (C) Summary plot illustrating that the initial firing frequency at the onset of a depolarizing current pulse is not altered by 4-AP (n = 10). (D) Summary plot showing that the final firing frequency of b-AC MCs is significantly decreased after bath application of 4-AP (n = 10). ^∗p < 0.05.

FIGURE 6

Emergence of slow afterdepolarizations (sADPs) in b-AC MCs in presence of 4-AP. (A) Representative response of a L2/3 b-AC MC to a 475 pA depolarizing current pulse before (black) and after (red) bath application of 4-AP. Following application of 4-AP, a sADP was observed following repetitive firing. (B) Summary plot showing that a significant s-ADP was observed in all b-AC MCs in presence of 4-AP (n = 10). *p < 0.05.

FIGURE 7

Effect of I_h inhibition with ZD 7288 on magnitude of evoked depolarizing GABAergic potentials in MCs. (A) Superimposition of representative responses of a L2/3 MC to a 250 pA hyperpolarizing current pulse. Characteristic sag (arrow) and rebound responses (arrow head) observed under control conditions (black) were inhibited following application of ZD 7288 (red). (B) Representative responses of evoked depolarizing GABAergic potentials in a L2/3 MC before (black) and after (red) bath application of ZD 7288. (C) Summary graph showing a significant increase in the amplitude of evoked depolarizing GABAergic potentials in MCs upon inhibition of I_h. (D) Summary plot indicating that ZD 7288 significantly increases the area of evoked depolarizing GABAergic potential responses in MCs (n = 7). ^∗p < 0.05.

FIGURE 8

Frequency and area of spontaneous depolarizing GABAergic potentials in L2/3 MCs increase with inhibition of I_h. (A) Specimen records showing spontaneous depolarizing GABAergic potentials before (black) and after (red) bath application of ZD 7288. A significant increase in the frequency of depolarizing GABAergic potentials was observed following inhibition of I_h. (B) An expanded view of a single spontaneous depolarizing GABAergic potential before (black) and after (red) bath application of ZD 7288. (C) Summary plot demonstrating a significant increase in the frequency of spontaneous GABAergic events following application of ZD 7288 (n = 9). (D) Summary graph demonstrating that inhibition of I_h significantly increases the area of spontaneous depolarizing GABAergic potentials. (n = 5). ^∗p < 0.05.

FIGURE 9

Evoked depolarizing GABAergic potentials are enhanced in FS-BCs following I_h inhibition. (A) Superimposition of representative responses of a L2/3 FS-BC to a 250 pA hyperpolarizing current pulse. Characteristic small amplitude sag (arrow) responses and rebound depolarizations (arrow head) were observed in FS-BCs under control conditions (black). Sag and rebound responses were blocked in presence of ZD 7288 (red). (B) Superimposed specimen records of evoked depolarizing GABAergic potentials in a L2/3 FS-BC before (black) and after (red) bath application of ZD 7288. (C) Summary plot demonstrating a significant increase in the amplitude of evoked depolarizing GABAergic potentials in FS-BCs upon I_h inhibition (n = 6). (D) Summary plot demonstrating ZD 7288 significantly increases the area of evoked depolarizing GABAergic potential responses in FS-BCs (n = 6). ^∗p < 0.05.

FIGURE 10

Frequency and area of spontaneous depolarizing GABAergic potentials in L2/3 FS-BCs increase with I_h inhibition. (A) Representative trace of a spontaneous depolarizing GABAergic potential (arrow) in a L2/3 FS-BC. Specimen record also shows characteristic 4-AP induced baseline burst firing in FS-BC, which precede and follow spontaneous depolarizing GABAergic potentials. (B) Representative trace of spontaneous events following bath application of ZD 7288. (C) Summary plot demonstrates that inhibition of I_h with ZD 7288 significantly increases the frequency of spontaneous depolarizing GABAergic potentials in FS-BCs (n = 7). (D) Summary plot demonstrates that the area of spontaneous depolarizing GABAergic potentials significantly increases with inhibition of I_h (n = 7). ^∗p < 0.05.

FIGURE 11

Changes in temporal integration of depolarizing IPSP in MCs and FS-BCs after I_h inhibition. (A) Ten superimposed responses of a L2/3 MC to a train of five stimuli at 25 Hz (20–50 μA) before (top, black) and after (bottom, red) bath application of Z7288. Temporal integration of IPSPs was observed under control conditions. This was significantly enhanced upon inhibition of I_h. (B) Superimposed averaged responses of the individual responses shown in (A). (C) Summary plot demonstrating ZD 7288 significantly increases in IPSP area in L2/3 MCs (n = 7). (D) Superimposed individual responses of a L2/3 FS-BC to a similar 25 Hz train before (top, black) and after (bottom, red) bath application of ZD7288. Characteristic IPSP depression was observed in the control condition. (E) Averaged responses of the individual traces shown in (D). Alterations in IPSP depression was not observed in FS-BCs following inhibition of I_h. However, ZD 7288 did cause the appearance of a late depolarization that could initiate depolarizing GABAergic potentials. (F) Summary plot illustrating that bath application of ZD 7288 significantly increases the area of depolarization after 25 Hz stimulation (n = 7). ^∗p < 0.05.