Chi-Kun Tong

NMDA receptors are important elements in pain signaling in the spinal cord dorsal horn. They are heterotetramers, typically composed of two GluN1 and two of four GluN2 subunits: GluN2A-2D. Mice lacking some of the GluN2 subunits show deficits in pain transmission yet functional synaptic localization of these receptor subtypes in the dorsal horn has not been fully resolved. In this study, we have investigated the composition of synaptic NMDA receptors expressed in monosynaptic and polysynaptic pathways from peripheral sensory fibers to lamina I neurons in rats. We focused on substance P receptor-expressing (NK1R+) projection neurons, critical for expression of hyperalgesia and allodynia. EAB-318 and (R)-CPP, GluN2A/B antagonists, blocked both monosynaptic and polysynaptic NMDA EPSCs initiated by primary afferent activation by ∼90%. Physiological measurements exploiting the voltage dependence of monosynaptic EPSCs similarly indicated dominant expression of GluN2A/B types of synaptic NMDA receptors. In addition, at synapses between C fibers and NK1R+ neurons, NMDA receptor activation initiated a secondary, depolarizing current. Ifenprodil, a GluN2B antagonist, caused modest suppression of monosynaptic NMDA EPSC amplitudes, but had a widely variable, sometimes powerful, effect on polysynaptic responses following primary afferent stimulation when inhibitory inputs were blocked to mimic neuropathic pain. We conclude that GluN2B subunits are moderately expressed at primary afferent synapses on lamina I NK1R+ neurons, but play more important roles for polysynaptic NMDA EPSCs driven by primary afferents following disinhibition, supporting the view that the analgesic effect of the GluN2B antagonist on neuropathic pain is at least in part, within the spinal cord.

Background: NMDA receptors expressed by spinal cord neurons in the superficial dorsal horn are involved in the development of chronic pain associated with inflammation and nerve injury. The superficial dorsal horn has a complex and still poorly understood circuitry that is mainly populated by inhibitory and excitatory interneurons. Little is known about how NMDA receptor subunit composition, and therefore pharmacology and voltage dependence, varies with neuronal cell type. NMDA receptors are typically composed of two NR1 subunits and two of four NR2 subunits, NR2A-2D. We took advantage of the differences in Mg²⁺sensitivity of the NMDA receptor subtypes together with subtype preferring antagonists to identify the NR2 subunit composition of NMDA receptors expressed on lamina II inhibitory and excitatory interneurons. To distinguish between excitatory and inhibitory interneurons, we used transgenic mice expressing enhanced green fluorescent protein driven by the GAD67 promoter.Results: Analysis of conductance ratio and selective antagonists showed that lamina II GABAergic interneurons express both the NR2A/B containing Mg²⁺sensitive receptors and the NR2C/D containing NMDA receptors with less Mg²⁺sensitivity. In contrast, excitatory lamina II interneurons express primarily NR2A/B containing receptors. Despite this clear difference in NMDA receptor subunit expression in the two neuronal populations, focally stimulated synaptic input is mediated exclusively by NR2A and 2B containing receptors in both neuronal populations.Conclusions: Stronger expression of NMDA receptors with NR2C/D subunits by inhibitory interneurons compared to excitatory interneurons may provide a mechanism to selectively increase activity of inhibitory neurons during intense excitatory drive that can provide inhibitory feedback.

NMDA receptors are important elements in pain signaling in the spinal cord dorsal horn. They are heterotetramers typically composed of two NR1 and two of four NR2 subunits: NR2A-2D. Mice lacking specific NR2 subunits show deficits in pain transmission yet subunit location in the spinal cord remains unclear. We have combined electrophysiological and pharmacological approaches to investigate the composition of functional NMDA receptors expressed by lamina I, substance P receptor-expressing (NK1R+) neurons, as well as NK1R- neurons. Under low Mg²⁺ conditions (100 μM), the conductance of NMDA receptors at −90 mV (g(−90 mV)) with NR2A or NR2B subunits (NR2A/B) is low compared to conductance measured at the membrane potential where the inward current is maximal or maximal inward current (MIC) (ratio of ~0.07 calculated from Kuner and Schoepfer, 1996). For NR2C or NR2D subunits (NR2C/D), the ratio is higher (ratio ~0.4). NK1R+ and NK1R- neurons express NMDA receptors that give ratios ~0.28 and 0.16, respectively, suggesting both types of subunits are present in both populations of neurons, with NK1R+ neurons expressing a higher percentage of NR2C/D type NMDA receptors. This was confirmed using EAB318, an NR2A/B preferring antagonist, and UBP141, a mildly selective NR2C/D antagonist to increase and decrease the g(−90 mV)/g(MIC) ratios in both subpopulations of neurons.

Blockade of Ca²⁺‐permeable AMPA receptors in the rat spinal cord diminishes the development of hyperalgesia and allodynia associated with peripheral injury. Cobalt uptake studies reveal that Ca²⁺‐permeable AMPA receptors are expressed by some substance P receptor‐expressing (NK1R+) neurons in lamina I, as well as other neurons throughout the superficial dorsal horn. Selective elimination of NK1R+ neurons in lamina I and lamina III/IV of the dorsal horn also suppresses development of hyperalgesia and allodynia. These observations raise the possibility that Ca²⁺‐permeable AMPA receptors contribute to excitatory synaptic drive onto the NK1R+ neurons associated with allodynia and hyperalgesia. The first synapse in the pain pathway is the glutamatergic excitatory drive from the primary afferent fibres onto dorsal horn neurons. Therefore, we tested whether Ca²⁺‐permeable AMPA receptors are located on lamina I and lamina III/IV NK1R+ neurons postsynaptic to primary afferent fibres, using inward rectification and polyamine toxins for receptor identification. We examined three different populations of dorsal horn neurons; lamina I NK1R+ neurons, including projection neurons, and non‐NK1R+ (NK1R–) neurons including interneurons, and lamina III/IV NK1R+ neurons, believed to contribute to the low‐threshold mechanosensory pathway. The majority of synapses in all three groups had rectification indices less than 1.0 and greater than 0.4, indicating that the AMPA receptors at these synapses are a mixture of Ca²⁺‐permeable and ‐impermeable forms. Lamina III/IV NK1R+ neurons and lamina I NK1R– neurons have a significantly higher proportion of postsynaptic Ca²⁺‐permeable AMPA receptors than lamina I NK1R+ neurons. Thus synaptically positioned Ca²⁺‐permeable AMPA receptors directly contribute to low‐threshold sensory afferent drive into the dorsal horn, and can mediate afferent input onto interneurons such as GABAergic neurons. These receptors also contribute to high‐threshold primary afferent drive onto NK1R+ neurons in the superficial dorsal horn, but do so less consistently.

The kinetics of activity-dependent, extracellular alkaline transients, and the buffering of extracellular pH (pH_e), were studied in rat hippocampal slices using a fluorescein-dextran probe. Orthodromic stimuli generated alkaline transients ≤0.05 pH units that peaked in 273 ± 26 ms and decayed with a half-time of 508 ± 43 ms. Inhibition of extracellular carbonic anhydrase (ECA) with benzolamide increased the rate of rise by 25%, doubled peak amplitude, and prolonged the decay three- to fourfold. The slow decay in benzolamide allowed marked temporal summation, resulting in a severalfold increase in amplitude during long stimulus trains. Addition of exogenous carbonic anhydrase reduced the rate of rise, halved the peak amplitude, but had no effect on the normalized decay. A simulation of extracellular buffering kinetics generated recoveries from a base load consistent with the observed decay of the alkaline transient in the presence of benzolamide. Under control conditions, the model approximated the observed decays with an acceleration of the CO₂hydration–dehydration reactions by a factor of 2.5. These data suggest low endogenous ECA activity, insufficient to maintain equilibrium during the alkaline transients. Disequilibrium implies a time-dependent buffering capacity, with a CO₂/HCO₃⁻contribution that is small shortly after a base load. It is suggested that within 100 ms, extracellular buffering capacity is about 1% of the value at equilibrium and is provided mainly by phosphate. Accordingly, in the time frame of synaptic transmission, small base loads would generate relatively large changes in interstitial pH.