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Relationship between tonic inhibitory currents and phasic inhibitory activity in the spinal cord lamina II region of adult mice
© Ataka and Gu; licensee BioMed Central Ltd. 2006
- Received: 01 November 2006
- Accepted: 27 November 2006
- Published: 27 November 2006
Phasic and tonic inhibitions are two types of inhibitory activities involved in inhibitory processing in the CNS. In the spinal cord dorsal horn, phasic inhibition is mediated by both GABAergic and glycinergic inhibitory postsynaptic currents. In contrast to phasic inhibitory currents, using patch-clamp recording technique on spinal cord slices prepared from adult mice we revealed that tonic inhibitory currents were mediated by GABAA receptors but not by glycine receptors in dorsal horn lamina II region. We found that there was a linear relationship (r = 0.85) between the amplitude of tonic inhibitory currents and the frequency of GABAergic inhibitory postsynaptic currents. Analysis of charge transfer showed that the charges carried by tonic inhibitory currents were about 6 times of charges carried by phasic inhibitory currents. The prominent charge transfer by tonic inhibitory currents and their synaptic activity dependency suggest a significant role of tonic inhibition in sensory processing.
- Dorsal Horn
- GABAA Receptor
- Glycine Receptor
- Spinal Cord Dorsal Horn
- Spinal Cord Slice
GABA (γ-Aminobutyric acid) and glycine are two principle inhibitory neurotransmitters in the spinal cord dorsal horn. They are either released separately or co-released from presynaptic terminals of inhibitory neurons. Upon the binding to GABAA receptors and glycine receptors at postsynaptic membrane, they elicit inhibitory postsynaptic currents (IPSCs). IPSCs provide phasic inhibition in neuronal network and are important for information processing. In addition to its action at synaptic sites, recent studies in several brain regions of matured animals have indicated that low concentrations of ambient GABA can activate high affinity GABAA receptors that are expressed at extrasynaptic sites to elicit a sustained inhibitory current [1–5]. A term 'tonic inhibitory currents' has been used to describe this sustained inhibitory current . Functionally, tonic GABAergic inhibition has been shown to control neuronal excitability in the brain [7–9]. Tonic inhibitory currents have been identified in rat cerebellar granule cells , granule cells of the dentate gyrus , thalamocortical relay neurons of the ventral basal complex , layer V pyramidal neurons in the somatosensory cortex , inhibitory interneurons in the CA1 region of the hippocampus . However, not all CNS neurons that were examined displayed tonic inhibitory currents under normal conditions. For example tonic inhibitory currents were normally not observed in hippocampal pyramidal cells in brain slices from adult animals [[5, 10], but see ].
The lamina II of the spinal dorsal horn (substantia gelatinosa) plays an important role in processing nociceptive input from fine myelinated Aδ – and unmyelinated C-primary afferents from the periphery . In this area, inhibitory neurons produce feedback and feed-forward inhibition to control nociceptive input, and a reduction of inhibitory activity in lamina II can result in central sensitization [13, 14]. Previous studies on inhibitory controls in the spinal cord dorsal horn have been mainly focused on inhibitory postsynaptic currents, i.e. phasic inhibition. Little is known about whether tonic inhibitory currents are present in this region and if so, whether tonic inhibitory currents are mediated by GABA receptors and/or glycine receptors.
The methods for preparing thick adult mouse spinal cord slices, as well as blind whole-cell patch-clamp recording techniques, have been described previously . In brief, transverse spinal cord slices (500–600 μm in thickness) were prepared from L5 spinal cords of adult mice (Harlan, IN, USA) aged between 6 and 9 weeks. In each experiment, a spinal cord slice was transferred to a recording chamber (volume of 0.5 ml). The slice was supported at the bottom by a nylon mesh in the recording chamber. The slice was superfused with Krebs solution at flow rate of 10 ml/min. The Krebs solution contained (in mM): NaCl 117, KCl 3.6, CaCl2 2.5, MgCl2, 1.2, NaH2PO4 1.2, NaHCO3 25, and glucose 11. The solution was equilibrated with 95% O2 and 5% CO2, maintained at room temperature (22°C), and the pH of the Krebs solution was 7.35. Under a dissecting microscope with 40× magnification, lamina regions were identified based on morphological features. The lamina II was clearly discernible as a relatively translucent band across the superficial dorsal horn. Under visual guidance, the patch electrode was inserted vertically into the lamina II.
Whole-cell patch-clamp recordings were made from lamina II neurons with electrodes (5~10 MΩ) filled with an internal solution containing (in mM): K-gluconate 120, KCl 20, MgCl2 2, Na2ATP 2, NaGTP 0.5, HEPES 20, EGTA 0.5, and pH 7.2 adjusted with NaOH. Signals were amplified and filtered at 2 kHz and sampled at 5 kHz (Axopatch 200B). Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded with cells being held at 0 mV. Each recording was performed on a cell in a fresh slice without prior application of any agonist or antagonist. Isolation of GABAergic sIPACs was accomplished by including 2 μM strychnine in the bath solution. Tonic inhibitory currents were revealed following the application of 20 μM bicuculline for a period of 3 min. All compounds tested were applied through the bath solution at a flow rate of 10 ml/min. Analysis of sIPSCs, including threshold setting and peak identification criteria, were performed according to a method previously described . Decay time constant (τ) of sISPCs was analyzed using Clampfit 9 (Axon Instruments, Inc., Sunnyvale, CA, USA). sIPSC frequency, amplitude, and average charge transfer (QsIPSCs, integrated area under sIPSCs) were analyzed using Mini Analysis Program (Synaptosft, Inc., Decatur, GA, USA). Charge transfer (QPC) associated with sIPSCs in a given time (t) was calculated using the equation QPC = f × QsIPSCs × t, where f is the frequencies (Hz) of sIPSCs, QsIPSCs is the average charge transfer per sIPSC during a 60-s baseline recording, and t is the duration (60 s), respectively. The charge transfer associated with tonic currents was calculated according to the equation: QTC = ITC × t, where QTC is the charge transfer produced by tonic currents, ITC is the current amplitude at steady-state, and t is time (60 s). Unless otherwise indicated, data represent Mean ± SEM, Student's t-tests were used for statistical comparison, and significance was considered at the P < 0.05 level.
Application of 20 μM bicuculline not only inhibited slow types of sIPSCs, but also caused a shift of baseline holding current (Figure 1B). The shift of baseline hold currents represents the presence of tonic inhibitory currents mediated by GABAA receptors . The amplitudes of the tonic inhibitory currents revealed by bicuculline were 8.4 ± 0.7 pA (n = 30, Figure 1D). After washing off bicuculline, hold currents returned to the baseline levels.
Glycinergic inhibitory postsynaptic currents contributed to phasic inhibitory activity in lamina II region, and application of glycine receptor antagonist strychnine (2 μM) blocked glycinergic sIPSCs. However, there was no significant change of baseline holding currents following the application 2 μM strychnine (Figure 1C). The baseline holding currents were -0.3 ± 0.5 pA (n = 6) in lamina II, not significantly different from the baseline noise level (Figure 1D). These results suggested that glycine receptors did not significantly account for the tonic inhibitory currents in dorsal horn lamina II neurons of adult mice.
Using spinal cord slice preparations from adult mice, the present study shows that tonic inhibitory currents are present in lamina II of the dorsal horn and was solely mediated by GABAA receptors, that the extent of tonic inhibition is proportional to GABAergic inhibitory synaptic activity, and that tonic currents transfer charges substantially higher than phasic currents. The results provide new information about inhibitory activities in a nociceptive processing region.
We have observed a large variation of the size of tonic inhibitory currents. It has been suggested that ambient GABA concentrations around the extrasynaptic domains of neurons is a factor that determines the size of tonic inhibitory currents. The concentrations of ambient GABA vary from tens of nanomolar to a few micromolar based on in vivo microdialysis studies in the brain [16–19]. One possible cause of the variation may be regional and temporal differences in GABAergic neuron activity. We have shown that GABAergic sIPSC frequencies recorded in lamina II neurons have a big variation, and GABAeric sIPSC frequency and tonic current size are correlated. It is very likely that neurons with higher GABAergic sIPSC frequency have higher concentrations of ambient GABA around them due to more frequent spillover of GABA from inhibitory synapses. Therefore, these neurons have larger-sized tonic inhibitory currents mediated by extrasynpatic GABAA receptors. This is consistent with previous studies in cerebellar granule cells, which shows that ambient GABA concentration is maintained by action potential-dependent vesicular release and is responsible for tonic GABAA receptor activation [1, 7, 20–23].
Although phasic inhibition is mediated by both GABAA and glycine receptors in spinal cord lamina II of adult mice, we did not observe any contribution of glycine receptors to tonic inhibitory currents. The lack of glycine receptor-mediated tonic inhibitory currents could be mainly due to the lack of high affinity glycine receptor isoforms. Of different glycine receptor isoforms (α1β, α2β, α3β) identified in the CNS, their EC50 values for glycine were normally above 50 μM . In contrast, extrasynaptic GABA receptors that contribute to tonic currents were found to be high affinity isoforms (e.g. α6βxδ, α4βxδ and α5βxγ2) with EC50 in nanomolar range .
The large charge transfer carried by GABAergic tonic inhibitory currents shown in this study indicates a persistent increase in the input conductance of lamina II dorsal horn neurons. The increase of input conductance in neurons can decrease the size and duration of the excitatory postsynaptic potentials (EPSPs), and make neurons less likely to generate action potentials. Indeed, tonic currents have been shown to be a critical determinant that controls neuron excitability in cerebellar granule cells [7–9]. In spinal cord lamina II, decreases of neuron excitability by GABAergic tonic inhibitory current may be an important mechanism to control nociceptive inputs and to prevent central hyper-sensitization.
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