Induction of long-term potentiation and long-term depression is cell-type specific in the spinal cord

Abstract

The underlying mechanism of chronic pain is believed to be changes in excitability in spinal dorsal horn (DH) neurons that respond abnormally to peripheral input. Increased excitability in pain transmission neurons, and depression of inhibitory neurons, are widely recognized in the spinal cord of animal models of chronic pain. The possible occurrence of 2 parallel but opposing forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD) was tested in 2 types of identified DH neurons using whole-cell patch-clamp recordings in mouse spinal cord slices. The test stimulus was applied to the sensory fibers to evoke excitatory postsynaptic currents in identified spinothalamic tract neurons (STTn) and GABAergic neurons (GABAn). Afferent conditioning stimulation (ACS) applied to primary afferent fibers with various stimulation parameters induced LTP in STTn but LTD in GABAn, regardless of stimulation parameters. These opposite responses were further confirmed by simultaneous dual patch-clamp recordings of STTn and GABAn from a single spinal cord slice. Both the LTP in STTn and the LTD in GABAn were blocked by an NMDA receptor antagonist, AP5, or an intracellular Ca chelator, BAPTA. Both the pattern and magnitude of intracellular Ca after ACS were almost identical between STTn and GABAn based on live-cell calcium imaging. The results suggest that the intense sensory input induces an NMDA receptor-dependent intracellular Ca increase in both STTn and GABAn, but produces opposing synaptic plasticity. This study shows that there is cell type-specific synaptic plasticity in the spinal DH.

Figure 1

High- or low-frequency afferent conditioning stimulation (ACS) (high-frequency conditioning stimulation [HFS] or low-frequency conditioning stimulation [LFS]) evokes long-term potentiation (LTP) in spinothalamic tract neurons (STTn) but long-term depression (LTD) in GABAn in the mouse spinal cord. (A) Frozen sections of the brain were made from mice injected with a tracer dye (DiI) and a composite drawing of DiI marking was made from 1 mouse as an example. DiI (red) spread in many areas of the brain but included the ventral posterior lateral (VPL) and ventral posterior medial (VPM) nuclei of the thalamus. (B and C) Examples of STTn identified by retrogradely labeled DiI (B) and GFP⁺ GABAn (C) that were patch-clamped for excitatory postsynaptic currents (EPSC) recordings (recording electrode edges are indicated by paired enclosing arrows). (D and E) Effects of a HFS (either 100 Hz for 1 second, repeated 3 times at a 10-second interval, holding potential at −50 mV or continuous 10-Hz pulses for 10 seconds with a holding potential of +10 mV) on EPSC amplitudes averaged from 11 STTn (D) and 11GABAn (E). The recording traces (shown above each plot) are the averages of 6 consecutive EPSC recordings before (a) and 15 minutes after (b) HFS. Calibration: 100 pA, 10 milliseconds. (F and G) Effects of an LFS (1 Hz for 100 seconds, holding potential −40 mV or 2-Hz pulses for 40 seconds, holding potential of +30 mV) on EPSC amplitudes averaged from 14 STTn (F) and 8 GABAn (G). Pie charts (shown above the plots in D–G) show the numbers of STT or GABA neurons that showed a significant change (>20%) after ACS.

Figure 2

An example of dual patch-clamp recordings of long-term potentiation in spinothalamic tract neurons (STTn) and long-term depression in GABAergic neurons (GABAn) from a single spinal cord slice. (A) A dual patch recording setup showing a DiI (red fluorescent)-labeled STTn (lower neuron) and GFP⁺ GABAn (upper neuron) with 2 patched recording electrodes (indicated by paired enclosing arrows) approached at a right angle. (B and C) Effects of 2-Hz afferent conditioning stimulation (ACS) (2 Hz for 40 seconds, holding potential +30 mV) on the amplitudes of excitatory postsynaptic currents (EPSCs) in STTn (B) and GABAn (C). The recording traces shown above the plots are averages of 6 consecutive EPSC recordings before (a) and 25 minutes after (b) the ACS. Calibration: 100 pA, 10 milliseconds.

Figure 3

Long-term potentiation (LTP) and long-term depression (LTD) induced by either electrical stimulation (ES) of the dorsal root or chemical stimulation (CS) of the spinal cord. (A–D) Induction of LTP and LTD by an electrical conditioning stimulation applied to the dorsal root. An attached dorsal root was suctioned into a glass stimulating electrode (A). Afferent conditioning stimulation (ACS) was delivered to the dorsal root, and the changes of the evoked excitatory postsynaptic current (EPSC) amplitudes were recorded from spinothalamic tract neurons (STTn) (B and C [n = 5]) and GABAergic neurons (GABAn) (B and D [n = 5]). The recording traces in B are averages of 6 consecutive EPSC recordings before and 15 minutes after ACS (calibration: 100 pA, 10 milliseconds). (E and F) Induction of synaptic plasticity in STTn and GABAn by a chemical conditioning stimulation. Spinal cord slices were treated with a mixture of 50 μM NMDA and 50 μM glycine for 5 minutes (indicated by a thick bar) while holding the membrane potential at −30 mV. The EPSC amplitudes were significantly increased in STTn while decreased in GABA neurons after NMDA/glycine treatment, suggesting the induction of LTP in STTn (E, n = 4) and LTD in GABA neurons (F, n = 6). Pie charts (shown above the plots in C–F) show the numbers of STT or GABA neurons that showed significant changes (>20%) after conditioning stimulations.

Figure 4

Induction, but not maintenance, of both long-term potentiation (LTP) in spinothalamic tract neurons (STTn) and long-term depression (LTD) in GABAergic neurons (GABAn) is NMDAR dependent. (A and B) The effect of pretreatment with an NMDAR blocker, AP5, on excitatory postsynaptic current (EPSC) amplitude in STTn (A, n = 7) and GABAn (B, n = 7). Spinal cord slices were superfused with AP5 (100 μM in ACSF) for the initial 15 minutes (only last 5 minutes is indicated by thick bars) during which baseline EPSCs were recorded, and then an afferent conditioning stimulation (ACS) was applied. The AP5 application was terminated immediately after the ACS application. Neither LTP in STTn nor LTD in GABAn was elicited by ACS after pretreatment with NMDA receptor antagonist. (C and D) The posttreatment effect of an NMDA receptor blocker, AP5, on EPSC amplitude in STTn (C, n = 6) and GABAn (D, n = 10). Spinal cord slices were superfused with AP5 (100 μM in ACSF) for at least 20 minutes (only first 15 minutes is indicated by thick bars) starting from 10 minutes after the ACS. Superfusion with AP5 produced no effect on already established LTP in STTn or LTD in GABAn.

Figure 5

Neither MCPG (nonselective mGluR antagonist) nor AP3 (mGluR 1 antagonist and inhibitor of phosphoserine phostphatase) had an effect on long-term depression (LTD) induction in spinal GABAergic neurons (GABAn). (A and B) Spinal cord slices were superfused with either MCPG (500 μM in ACSF) or AP3 (30 μM in ASCF) for 5 minutes during excitatory postsynaptic current (EPSC) baseline recordings, and then afferent conditioning stimulation (ACS) was applied. Neither MCPG (A, n = 5) nor AP3 (B, n = 4) had any effect on the induction of LTD in GABAn.

Figure 6

The induction of both long-term potentiation (LTP) in spinothalamic tract neurons (STTn) and long-term depression (LTD) in GABAergic neurons (GABAn) share the same mechanism of NMDAR-mediated rapid increase in intracellular Ca²⁺ ([Ca²⁺]_i). (A and B)[Ca²⁺]_i levels were visualized with Oregon Green 488 BAPTA-1 (0.2 mM, administered intracellularly by preloading it into the patch pipette) before, during, and after 2-Hz ACS in an STTn and a GABAn (A). The averaged changes of [Ca²⁺]_i levels after 2-Hz ACS (B) show that intracellular free-Ca²⁺ levels in both STTn and GABAn increase at a similar rate and amplitude (n = 4 each). (C and D) Afferent conditioning stimulation with 2 Hz failed to induce either LTP in spinothalamic tract neurons (STTn) (C) or LTD in GABAn (D) when Ca²⁺ chelator, BAPTA (5 mM), was administered intracellularly by preloading it into the patch pipette.