The heterogeneity in GABAA receptor-mediated IPSC kinetics reflects heterogeneity of subunit composition among inhibitory and excitatory interneurons in spinal lamina II

GABAergic inhibition displays rich functional diversity throughout the CNS, which arises from variations in the nature of inputs, subunit composition, subcellular localization of receptors and synapse geometry, or reuptake mechanisms. In the spinal dorsal horn (SDH), GABAA and glycine receptors play a major role in the control of excitability and accuracy of nociceptive processing. Identifying which components shape the properties of the inhibitory synapses in different cell types is necessary to understand how nociceptive information is integrated. To address this, we used transgenic mice where inhibitory interneurons express GAD65-EGFP. We found that GABAA, but not glycine receptor-mediated evoked IPSCs displayed slower kinetics in EGFP+ vs. EGFP− interneurons. GABAA miniature IPSC decay kinetics showed a large variability in both populations, however the distribution of decays differed between EGFP+ and EGFP− interneurons. The range of mIPSC decay kinetics observed was replicated in experiments using rapid application of GABA on outside-out patches taken from SDH neurons in slices. Furthermore, GABAA decay kinetics were not affected by uptake blockers and were not different in mice lacking δ or α5 subunits, indicating that intrinsic channel properties likely underlie the heterogeneity. To identify whether other α subunits shape the various kinetic properties observed we took advantage of knock-in mice carrying point mutations in either the α1, α2, or α3 subunits rendering Ro 15-4513 a selective agonist at the benzodiazepine modulatory site. We found that α1 and α2 subunit underlie the fast decaying component of IPSCs while the slow component is determined by the α3 subunit. The differential distribution of GABAA subunits at inhibitory synapses thus sculpts the heterogeneity of the SDH inhibitory circuitry. This diversity of inhibitory elements can be harnessed to selectively modulate different components of the spinal nociceptive circuitry for therapeutic interventions

Kinetics of ATP release following compression injury of a peripheral nerve trunk

Compression and/or contusion of a peripheral nerve trunk can result in painful sensations. It is possible that release of ATP into the extracellular space may contribute to this symptom. In the present study, we used real-time measurements of ATP-induced bioluminescence together with electrophysiological recordings of compound action potentials to follow changes in the extracellular ATP concentration of isolated rat spinal roots exposed to mechanical stimuli. Nerve compression for about 8 s resulted in an immediate release of ATP into the extracellular space and in a decrease in the amplitude of compound action potentials. On average, a rise in ATP to 60 nM was observed when nerve compression blocked 50% of the myelinated axons. After the compression, the extracellular concentration of ATP returned to the resting level within a few minutes. The importance of ecto-nucleotidases for the recovery period was determined by exposure of isolated spinal roots to high concentrations of ATP and by use of inhibitors of ecto-nucleotidases. It was observed that spinal roots have a high capacity for ATP hydrolysis which is only partially blocked by βγ-methylene ATP and ARL 67156. In conclusion, acute nerve compression produces an increase in the extracellular concentration of ATP and of its metabolites which may be sufficient for activation of purinergic P2 and/or P1 receptors on axons of nociceptive afferent neurons

GABA-A Channel Subunit Expression in Human Glioma Correlates with Tumor Histology and Clinical Outcome

GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the CNS and is present in high concentrations in presynaptic terminals of neuronal cells. More recently, GABA has been ascribed a more widespread role in the control of cell proliferation during development where low concentrations of extrasynaptic GABA induce a tonic activation of GABA receptors. The GABA-A receptor consists of a ligand-gated chloride channel, formed by five subunits that are selected from 19 different subunit isoforms. The functional and pharmacological properties of the GABA-A channels are dictated by their subunit composition. Here we used qRT-PCR to compare mRNA levels of all 19 GABA-A channel subunits in samples of human glioma (n = 29) and peri-tumoral tissue (n = 5). All subunits except the ρ1 and ρ3 subunit were consistently detected. Lowest mRNA levels were found in glioblastoma compared to gliomas of lower malignancy, except for the θ subunit. The expression and cellular distribution of the α1, γ1, ρ2 and θ subunit proteins was investigated by immunohistochemistry on tissue microarrays containing 87 gliomas grade II. We found a strong co-expression of ρ2 and θ subunits in both astrocytomas (r = 0.86, p<0.0001) and oligodendroglial tumors (r = 0.66, p<0.0001). Kaplan-Meier analysis and Cox proportional hazards modeling to estimate the impact of GABA-A channel subunit expression on survival identified the ρ2 subunit (p = 0.043) but not the θ subunit (p = 0.64) as an independent predictor of improved survival in astrocytomas, together with established prognostic factors. Our data give support for the presence of distinct GABA-A channel subtypes in gliomas and provide the first link between specific composition of the A-channel and patient survival

Synaptic Connections of the Neurokinin 1 Receptor-Like Immunoreactive Neurons in the Rat Medullary Dorsal Horn

The synaptic connections between neurokinin 1 (NK1) receptor-like immunoreactive (LI) neurons and γ-aminobutyric acid (GABA)-, glycine (Gly)-, serotonin (5-HT)- or dopamine-β-hydroxylase (DBH, a specific marker for norepinephrinergic neuronal structures)-LI axon terminals in the rat medullary dorsal horn (MDH) were examined under electron microscope by using a pre-embedding immunohistochemical double-staining technique. NK1 receptor-LI neurons were observed principally in laminae I and III, only a few of them were found in lamina II of the MDH. GABA-, Gly-, 5-HT-, or DBH-LI axon terminals were densely encountered in laminae I and II, and sparsely in lamina III of the MDH. Some of these GABA-, Gly-, 5-HT-, or DBH-LI axon terminals were observed to make principally symmetric synapses with NK1 receptor-LI neuronal cell bodies and dendritic processes in laminae I, II and III of the MDH. The present results suggest that neurons expressing NK1 receptor within the MDH might be modulated by GABAergic and glycinergic inhibitory intrinsic neurons located in the MDH and 5-HT- or norepinephrine (NE)-containing descending fibers originated from structures in the brainstem