GABAA and Glycine Receptor-Mediated Inhibitory Synaptic Transmission onto Adult Rat Lamina IIi PKCγ-Interneurons: Pharmacological but Not Anatomical Specialization

Abstract

Mechanical allodynia (pain to normally innocuous tactile stimuli) is a widespread symptom of inflammatory and neuropathic pain. Spinal or medullary dorsal horn (SDH or MDH) circuits mediating tactile sensation and pain need to interact in order to evoke mechanical allodynia. PKCγ-expressing (PKCγ⁺) interneurons and inhibitory controls within SDH/MDH inner lamina II (II_i) are pivotal in connecting touch and pain circuits. However, the relative contribution of GABA and glycine to PKCγ⁺ interneuron inhibition remains unknown. We characterized inhibitory inputs onto PKCγ⁺ interneurons by combining electrophysiology to record spontaneous and miniature IPSCs (sIPSCs, mIPSCs) and immunohistochemical detection of GABA_ARα2 and GlyRα1 subunits in adult rat MDH. While GlyR-only- and GABA_AR-only-mediated mIPSCs/sIPSCs are predominantly recorded from PKCγ⁺ interneurons, immunohistochemistry reveals that ~80% of their inhibitory synapses possess both GABA_ARα2 and GlyRα1. Moreover, nearly all inhibitory boutons at gephyrin-expressing synapses on these cells contain glutamate decarboxylase and are therefore GABAergic, with around half possessing the neuronal glycine transporter (GlyT2) and therefore being glycinergic. Thus, while GABA and glycine are presumably co-released and GABA_ARs and GlyRs are present at most inhibitory synapses on PKCγ⁺ interneurons, these interneurons exhibit almost exclusively GABA_AR-only and GlyR-only quantal postsynaptic inhibitory currents, suggesting a pharmacological specialization of their inhibitory synapses.

Keywords: gephyrin; glutamic acid decarboxylase; glycine transporter 2; inhibitory interneurons; mechanical allodynia; medullary dorsal horn; miniature IPSCs; protein kinase C gamma; spontaneous IPSCs; α1 subunit of glycine receptor; α2 subunit of GABAA receptor.

Figure 1

Functional properties of mIPSCs recorded from lamina II_i PKCγ⁺ interneurons. (A) PKCγ⁺ neurobiotin-filled interneuron. Confocal images showing the neurobiotin labeling of a recorded neuron (red, arrowhead) that colocalized with the PKCγ immunostaining (green; (A1)). Immuno-labeling was performed in parasagittal slices (350 µm thick). Representative neuronal reconstruction showing the neuritic arborization of recorded PKCγ⁺ interneuron (A2). M, medial; L lateral; D, dorsal; V, ventral. Dashed lines represent laminae limits. (B) (Top) Whole-cell patch-clamp recordings (voltage-clamp mode; holding potential: −65 mV) of miniature inhibitory postsynaptic currents (mIPSCs) from a PKCγ⁺ interneuron (in the presence of CNQX (10 μM) and APV (40 μM)). (Bottom) Magnified events of the three categories of mIPSCs (on the basis of their decay kinetics): left, slowly biexponentially decaying mIPSC (Biexpo); middle, fast monoexponentially decaying mIPSC (Fast monoexpo); right, slowly monoexponentially decaying mIPSC (Slow monoexpo). Each computed tau (τ) value is indicated close to the corresponding component. (C) Close inspection of the kinetics of the monoexponentially decaying mIPSC from PKCγ⁺ interneurons revealed a heterogeneous population of decay times, which were best fitted by the sum of two Gaussians. (D–F) Boxplots of the decay τ (D), the rise τ (E), and the amplitude (F) of the fast monoexponentially decaying, the slowly monoexponentially decaying, and the slowly biexponentially decaying mIPSCs; the τ values of the latter were obtained by forcing monoexponential fits. Data are presented as box-and-whisker plots depicting median, interquartile interval, minimum, and maximum. The “+” represents the medians, and the boxes present the quartiles. Data were averaged from 5 lamina II_i PKCγ⁺ interneurons. The comparison between groups was made using one-way ANOVA followed by Tukey’s post-hoc test. * p < 0.05.

Figure 2

Proportions of kinetically defined sIPSC and mIPSCs recorded from lamina IIi PKCγ⁺ and PKCγ⁻ interneurons. Bar histogram showing the mean ± SEM of proportions (%) of the different kinetically defined (fast monoexponential, slow monoexponential, and biexponential) spontaneous (sIPSC) or miniature inhibitory postsynaptic currents (mIPSC) recorded from lamina II_i PKCγ⁺ (n = 10) or PKCγ⁻ (n = 10) interneurons. The comparison between groups was made using two-way ANOVA followed by Tukey’s post-hoc test. * p < 0.05.

Figure 3

GlyR- and GABA_AR-mediated sIPSCs in lamina II_i PKCγ⁺ and PKCγ⁻ interneurons. (A) Whole-cell patch-clamp recordings (voltage-clamp mode; holding potential: −65 mV) of spontaneous inhibitory postsynaptic currents (sIPSCs) from a lamina II_i PKCγ⁺ interneuron in control condition (in the presence of CNQX (10 μM) and APV (40 μM)) and after application of bicuculline (Bic; GABA_AR antagonist, 10 μM) and strychnine (Stry; GlyR antagonist, 0.5 µM). Note that sIPSC were strongly reduced (number) after bicuculline and completely abolished after bicuculline + strychnine. (B) Cumulative probability plots of decay τ of sIPSCs recorded from 4 lamina II_i neurons, 2 PKCγ⁺ (black), and 2 PKCγ⁻ (gray) neurons. The plots were constructed by forcing monoexponential fits to all individual sIPSCs recorded under control conditions (solid lines) and then in the presence of bicuculline (dashed lines). Note that bicuculline abolished all events with decay τ higher than 20 ms.

Figure 4

Inhibitory receptors and synapses on lamina II_i PKCγ⁺ interneurons. (A) Confocal images showing a PKCγ⁺ interneuron (red) with GABA_ARα2 (green) and GlyRα1 (blue) labelings. The merged image shows that these inhibitory receptor subunits were colocalized on the cell body (arrowheads). Scale bar = 5 µm. (B) Proportions of PKCγ⁺ interneurons that display GABA_Aergic and/or glycinergic receptors subunits (n = 75). (C) Confocal image of a single optical section through a part of lamina II_i, scanned to reveal PKCγ (red), gephyrin (white), GlyT2 (green), and GAD (blue). Gephyrin labeling indicates the localization of inhibitory synapses on the PKCγ cell body, and GlyT2 and GAD labelings show the terminal synaptic boutons. The arrowhead indicates a synapse from a GAD-positive GlyT2-negative bouton, while the inset (corresponding to the box magnification, scale bar = 1 µm) shows a synapse from a bouton that is positive for both GAD and GlyT2 (scale bar = 5 µm).