Enhancement of excitatory synaptic integration by GABAergic inhibition in the subthalamic nucleus

Abstract

The activity patterns of subthalamic nucleus (STN) neurons, which are intimately related to normal movement and abnormal movement in Parkinson's disease (PD), are sculpted by feedback GABAergic inhibition from the reciprocally connected globus pallidus (GP). To understand the principles underlying the integration of GABAergic inputs, we used gramicidin-based patch-clamp recording of STN neurons in rat brain slices. Voltage-dependent Na+ (Nav) channels actively truncated synthetic IPSPs and were required for autonomous activity. In contrast, hyperpolarization-activated cyclic nucleotide-gated and class 3 voltage-dependent Ca2+ channels contributed minimally to the integration of single or low-frequency trains of IPSPs and autonomous activity. Interestingly, IPSPs modified action potentials (APs) in a manner that suggested IPSPs enhanced postsynaptic Nav channel availability. This possibility was confirmed in acutely isolated STN neurons using current-clamp recordings containing IPSPs as voltage-clamp waveforms. Tetrodotoxin-sensitive subthreshold and spike-associated Na+ currents declined during autonomous spiking but were indeed transiently boosted after IPSPs. A functional consequence of inhibition-dependent augmentation of postsynaptic excitability was that EPSP-AP coupling was dramatically improved when IPSPs preceded EPSPs. Because STN neuronal activity exhibits coherence with cortical beta-oscillations in PD, we tested how rhythmic sequences of cortical EPSPs were integrated in the absence and presence of feedback inhibition. STN neuronal activity was consistently entrained by EPSPs only in the presence of feedback inhibition. These observations suggest that feedback inhibition from the GP is critical for the emergence of coherent beta-oscillations between the cortex and STN in PD.

Figure 1.

Na_v channels pace autonomous activity and participate in inhibitory synaptic integration. Selective blockade of ∼47% Na_v channels with 5 nm TTX elevated APth and reduced the frequency of firing in a reversible manner (Ai–Aiii, individual example; Bi, Bii, population data; n = 7). Aiv, Complete blockade of Na_v channels abolished firing and led to a stable membrane potential below APth (inset in Aiv, sensitivity of peak Na⁺ current evoked by a 50 ms step from –90 to –45 mV to TTX in acutely isolated STN neurons; n = 6). The amplitude of dIPSPs measured at approximately –55 mV and approximately –65 mV was not altered, but their integral was increased by the application of saturating concentrations of TTX (Ci, representative examples at –54 and –64 mV; Cii–Ciii, population data; n = 9). D, A partial reduction in Na_v channel availability with 2–5 nm TTX affected the resetting of autonomous activity by GABA_A receptor-mediated dIPSPs. Di, Superimposition of 50 trials in control and low TTX conditions. The mean and SD of the latency of APs after the dIPSP for this example are shown at the top of each graph. Dii, Diii, Population data illustrating that a decrease in Na_v channel availability increases the latency (Dii) and reduces the precision (Diii; SD of latency) of APs generated after dIPSPs (n = 11). *p < 0.05. All recordings were performed in the presence of ionotropic glutamate and GABA receptor antagonists (50 μm APV, 20 μm DNQX, 20 μm GABAzine, and 1–2 μm CGP55845).

Figure 2.

HCN and Ca_v3 channels are not essential for autonomous activity and do not participate in the resetting of oscillations by GABAergic IPSPs. A, Impact of Cs⁺ and Ni²⁺ treatments on autonomous activity in STN neurons. Ai–Aiii, Individual examples of 5 s of spontaneous firing in control conditions (i), the absence of effect of Cs⁺ on autonomous firing (ii), and the increase in autonomous firing frequency in the presence of 50 μm Ni²⁺ (iii). Bi, Bii, Summary of the effect of Cs⁺ and Ni²⁺ on the frequency (i) and precision (CV; ii) of autonomous oscillation in six neurons. C, Blockade of HCN and Ca_v3 channels had no effect on the resetting of oscillations by GABA_A receptor-mediatedIPSPs.Ci–Ciii, Superimposition of 50 trials showing resetting by dIPSPs in control conditions (i), in the presence of Cs⁺ (ii), and in the presence of Cs⁺ and Ni²⁺ (iii). Time windows of AP generation after dIPSPs are depicted by gray rectangles. Di, Dii, Population data demonstrating that HCN and Ca_v3 channel blockade did not alter the latency (i) and precision (ii; SD of the latency) of APs generated after dIPSPs (n = 6).

Figure 3.

GABA_A IPSPs modify the morphologies of APs. A, Multiple electrically stimulated IPSPs (Ai) lowered threshold (Aii–Aiv) and increased the maximal speed (Aiii, Aiv) of APs generated after IPSPs. Aii, Overlaid APs generated before (AP–1) and after (AP 1, AP 2) multiple IPSPs. Aiii, Plots of (δV/δt)/V illustrate the method used to measure APth (defined as first point of sustained positive acceleration of voltage [(δV/δt)/δt] that was also >2 times the SD of membrane noise before APth) and the maximal speed [(δV/δt)max] of APs. Other characteristic features of APs on the phase plot, such as the spike after hyperpolarization (AHP) and the peak of the AP, are also noted. Inset, Zoom of APths (dots). Aiv, Population sample of the IPSP-induced changes in APth and maximal speeds of APs (n = 9). B, Single electrically stimulated IPSPs (Bi) lowered APth (Bii–Biv) and increased the maximal speed of APs (Biii, Biv) generated after an IPSP in the same neuron. Bii, Overlaid APs generated before (AP–1) and after(AP1)anIPSP.Biii, Phaseplot showing APth. Insets, Zoom of APths (dots). Biii, Population sample of the change in APth and maximal speed of APs (n = 9). Larger effects were observed in individual neurons (Ai–Aiii vs Bi–Biii) and across the population sample (Aiv vs Biv) after multiple IPSPs. C, Spontaneous IPSPs modified APths in a similar manner to electrically stimulated IPSPs or dIPSPs. Ci, Example of autonomous activity with (gray trace) and without (black trace; IPSPs blocked by GABAzine) spontaneous IPSPs in the same neuron. Cii, Overlaid APs in each condition. Inset, Mean APth in the presence and absence of spontaneous inhibition (n = 3). *p < 0.05.

Figure 4.

Inactivation of Na_v channels during autonomous activity is relieved in a duration-dependent manner by IPSPs. Autonomous spiking and inhibitory activity recorded using the perforated patch-clamp technique was replayed to acutely isolated neurons as voltage-clamp waveforms. This approach revealed that mean TTX-sensitive subthreshold and peak spike-associated currents declined during tonic spiking activity but were transiently increased after multiple (A) and single (B) IPSPs in representative neurons (Ai, Bi; average of 3 trials) and for the sample population (Aii, Aiii, n = 13; Bii, Biii, n = 16) compared with the mean currents flowing in the previous five oscillatory cycles. Expanded views (gray rectangles) of waveform and interspike current flowing before, during, and after IPSPs demonstrate that, during IPSPs, Na_v channel currents are reduced. After multiple (Aiii, n = 13) and single (Biii, n = 16) IPSPs, subthreshold current was significantly increased throughout the range of voltages traversed during the interspike interval. *p < 0.05. I, Current; V, voltage.

Figure 5.

The coupling of APs to EPSPs is more efficient when EPSPs are preceded by but are not coincident with dIPSPs. A–G, Comparison of APs generated over multiple trials after EPSPs (20 trials; Ai, Aii, D–G), EPSPs preceded by dIPSPs (20 trials; Bi, Bii, D–G), and coincident EPSPs and dIPSPs (10 trials; Ci, Cii, D–G) in a typical neuron; 0 ms represents the time at which the EPSP was electrically stimulated. Ai, Bi, Ci, Rasters of AP activity over multiple trials of the three protocols. The time windows over which APs were generated after EPSPs are highlighted by colored rectangles. Aii, Bii, Cii, Ten examples of APs generated in response to EPSPs for each protocol. D, Comparison of EPSPs and subsequent APs evoked at identical voltages in the absence of a dIPSP (red), after a dIPSP (blue), and with a coincident dIPSP (purple). E, APth was significantly lowered when EPSPs were preceded by dIPSPs. The reduction in APth was also significantly greater than for EPSPs preceded by dIPSPs than for dIPSPs alone (compare with Fig. 3C). F, G, The latency (F) and the variability (G; SD of latency) of AP generation after EPSPs were reduced when EPSPs were preceded by but not coincident with dIPSPs. Results for seven neurons (each neuron is represented by a distinct symbol and color) are represented graphically. Black horizontal bars represent the mean latency and mean SD of latency associated with each protocol in seven neurons. *p < 0.05.

Figure 6.

A, Comparison of APs generated over 50 trials after a dIPSP–dEPSP sequence in control conditions (Ai; black traces) and in 2 nm TTX, which reduced the availability of Na_v channels by ∼20% (Aii; gray traces); 0 ms represents the time at which the dEPSP was generated. B, The latency (Bi) and the variability (Bii; SD of latency) of AP generation after the dIPSP–dEPSP sequence were significantly increased in the presence of 2 nm TTX. Results for six neurons (each neuron is represented by a distinct symbol) are represented graphically. Black horizontal bars represent the mean latency and mean SD of latency associated with each experimental condition in six neurons. CTR, Control. Ci–Ciii, Examples of superimposed individual traces taken from the same neuron in control conditions (black traces) and in 2 nm TTX (gray traces) at various phases of the interspike interval. Note that the dEPSP-driven AP occurs earliest in control conditions, even when the membrane potential at which the dEPSP is injected is more hyperpolarized. *p < 0.05.

Figure 7.

Feedback inhibition enhances the coupling of APs to rhythmic EPSPs. A, Trains of electrically stimulated EPSPs did not drive synchronized spiking in STN neurons. Ai, Top, Twenty superimposed trials during which a train of EPSPs was evoked at a frequency of 14 Hz (gray rectangle) for a period of 1 s. EPSPs were stimulated at the times marked by the vertical bars above the graph. Bottom, Enlargement of EPSP-driven APs. Relatively weak phase-locked activity was observed in the peristimulus raster plot (Aii, highlighted gray box) and the peristimulus time histogram (Aiii) during the period of evoked EPSPs. B, Synthetic feedback inhibition enhanced EPSP-driven APs and promoted the emergence of phase-locked neuronal activity. Bi, Top, Overlayof20trialsinwhichEPSPsandfeedbackdIPSPsweregeneratedatafrequencyof14 Hz. Each dIPSP was injected 20 ms after each evoked EPSP for 1 s (protocol illustrated above the graph). Bottom, Enlargement of excitation–inhibition sequences revealing the precise phase locking of EPSP-driven APs when feedback inhibition was incorporated. Aligned APs were more apparent on the peristimulus raster plots (Bii, gray box) and the peristimulus time histogram (Biii). C, Comparison of EPSPs and subsequent APs evoked at identical voltages in the absence (red) and presence (blue) of feedback inhibition. D, The latency of AP generation was reduced and its precision (SD of latency) improved when dIPSPs intervened evoked EPSPs (blue) compared with the stimulation of EPSPs in isolation (red).

Figure 8.

Feedback inhibition enhances the coupling of APs to rhythmic dEPSPs. Ai, Failure of an injected train of dEPSPs to drive phase-locked spiking in an STN neuron. Top, Twenty superimposed trials in which a train of dEPSPs was injected at a frequency of 18 Hz (gray rectangle) for a period of 1 s. The pattern of dEPSP injection is shown above the graph. Bottom, Enlargement showing the imprecise timing of APs in response to rhythmic dEPSPs. The increase in stimulus-evoked neuronal activity on the peristimulus raster plot (Aii, highlighted gray box) and the peristimulus time histogram (Aiii) were not accompanied by correlated activity. B, Feedback dIPSPs enhance the coupling dEPSPs and APs, leading to the emergence of synchronized neuronal activity. Bi, Top, Overlay of 20 trials in which dEPSPs and dIPSPs were generated at a frequency of 18 Hz. Each dIPSP was injected 10 ms after the dEPSP, and this sequence was repeated for 1 s (protocol illustrated above the graph). Bi, Bottom, Enlargement of excitation–inhibition sequences revealing the phase locking of APs to dEPSPs in the presence of feedback inhibition. Precisely driven APs were apparent from the peristimulus raster plot (Bii, gray box) and the peristimulus time histogram (Biii). C, Comparison of dEPSPs and subsequent APs evoked at identical voltages in the absence (red) and presence (blue) of feedback inhibition. D, The latency of AP generation was reduced and its precision (SD of latency) was improved when feedback inhibition was incorporated (blue) compared with rhythmic excitation alone (red).