A phospho-deficient α3 glycine receptor mutation alters synaptic glycine and GABA release in mouse spinal dorsal horn neurons

Glycine receptors (GlyRs), together with GABA$_{A}$ receptors, mediate postsynaptic inhibition in most spinal cord and hindbrain neurons. In several CNS regions, GlyRs are also expressed in presynaptic terminals. Here, we analysed the effects of a phospho-deficient mutation (S346A) in GlyR α3 subunits on inhibitory synaptic transmission in superficial spinal dorsal horn neurons, where this subunit is abundantly expressed. Unexpectedly, we found that not only were the amplitudes of evoked glycinergic inhibitory postsynaptic currents (IPSCs) significantly larger in GlyRα3(S346A) mice than in mice expressing wild-type α3GlyRs (GlyRα3(WT) mice), but so were those of GABAergic IPSCs. Decreased frequencies of spontaneously occurring glycinergic and GABAergic miniature IPSCs (mIPSCs) with no accompanying change in mIPSC amplitudes suggested a change in presynaptic transmitter release. Paired-pulse experiments on glycinergic IPSCs revealed an increased paired-pulse ratio and a smaller coefficient of variation in GlyRα3(S346A) mice, which together indicate a reduction in transmitter release probability and an increase in the number of releasable vesicles. Paired-pulse ratios of GABAergic IPSCs recorded in the presence of strychnine were not different between genotypes, while the coefficient of variation was smaller in GlyRα3(S346A) mice, demonstrating that the decrease in release probability was readily reversible by GlyR blockade, while the difference in the size of the pool of releasable vesicles remained. Taken together, our results suggest that presynaptic α3 GlyRs regulate synaptic glycine and GABA release in superficial dorsal horn neurons, and that this effect is potentially regulated by their phosphorylation status. KEY POINTS: A serine-to-alanine point mutation was introduced into the glycine receptor α3 subunit of mice. This point mutation renders α3 glycine receptors resistant to protein kinase A mediated phosphorylation but has otherwise only small effects on receptor function. Patch-clamp recordings from neurons in mouse spinal cord slices revealed an unexpected increase in the amplitudes of both glycinergic and GABAergic evoked inhibitory postsynaptic currents (IPSCs). Miniature IPSCs, paired-pulse ratios and synaptic variation analyses indicate a change in synaptic glycine and GABA release. The results strongly suggest that α3 subunit-containing glycine receptors are expressed on presynaptic terminals of inhibitory dorsal horn neurons where they regulate transmitter release

3D Surface Measurement for Medical Application—Technical Comparison of Two Established Industrial Surface Scanning Systems

In 3D mapping of flexible surfaces (e.g. human faces) measurement errors due to movement or positioning occur. Aggravated by equipment- or researcher-caused mistakes considerable deviations can result. Therefore first the appliances' precision handling and reliability in clinical environment must be established. Aim of this study was to investigate accuracy and precision of two contact-free 3D measurement systems (white light vs. laser). Standard specimens of known diameter for sphere deviation, touch deviation and plane deviation were tested. Both systems are appropriate for medical application acquiring solid data (<mm). The more complex white-light system shows better accuracy at 0.2s measuring time. The laser system is superior concerning robustness, while accuracy is poorer and input time (1.5-2.5s) longer. Due to the clinical demand the white-light system is superior in a laboratory environment, while the laser system is easier to handle under non-laboratory condition

Efficient and reliable finite element methods for simulation of the human mandible

By computed tomography data (CT), the individual geometry of the mandible is quite well reproduced, also the seperation of cortical and trabecular bone. Using anatomical knowledge about the architecture and the functional potential of the masticatory muscles realistic situations can be approximated. The solution of the underlying partial differential equations describing linear elastic material behaviour is provided by an adaptive finite element method. Estimates of the discretization errors, local grid refinement, and multilevel technique guarantee the reliability and efficiency of the method

Inhibitory Kcnip2 neurons of the spinal dorsal horn control behavioral sensitivity to environmental cold

Proper sensing of ambient temperature is of utmost importance for the survival of euthermic animals, including humans. While considerable progress has been made in our understanding of temperature sensors and transduction mechanisms, the higher-order neural circuits processing such information are still only incompletely understood. Using intersectional genetics in combination with circuit tracing and functional neuron manipulation, we identified Kcnip2-expressing inhibitory (Kcnip2GlyT2) interneurons of the mouse spinal dorsal horn as critical elements of a neural circuit that tunes sensitivity to cold. Diphtheria toxin-mediated ablation of these neurons increased cold sensitivity without affecting responses to other somatosensory modalities, while their chemogenetic activation reduced cold and also heat sensitivity. We also show that Kcnip2GlyT2 neurons become activated preferentially upon exposure to cold temperatures and subsequently inhibit spinal nociceptive output neurons that project to the lateral parabrachial nucleus. Our results thus identify a hitherto unknown spinal circuit that tunes cold sensitivity. Keywords: circuit; cold; cold allodynia; cold analgesia; cooling; dre recombinase; interneuron; intersectional gene targeting; kcnip2; pai

Three-dimensional cephalometric evaluation of maxillary growth following in utero repair of cleft lip and alveolar-like defects in the mid-gestational sheep model

Objective: To evaluate maxillary growth following in utero repair of surgically created cleft lip and alveolar (CLA)-like defects by means of three-dimensional (3D) computer tomographic (CT) cephalometric analysis in the mid-gestational sheep model. Methods: In 12 sheep fetuses a unilateral CLA-like defect was created in utero (untreated control group: 4 fetuses). Four different bone grafts were used for the alveolar defect closure. After euthanasia, CT scans of the skulls of the fetuses, 3D re-constructions, and a 3D-CT cephalometric analysis were performed. Results: The comparisons between the operated and nonoperated skull sides as well as of the maxillary asymmetry among the experimental groups revealed no statistically significant differences of the 12 variables used. Conclusions: None of the surgical approaches used for the in utero correction of CLA-like defects seem to affect significantly postsurgical maxillary growth; however, when bone graft healing takes place, a tendency for almost normal maxillary growth can be observed. Copyright (c) 2006 S. Karger AG, Basel

Antihyperalgesia by α2-GABAA Receptors Occurs Via a Genuine Spinal Action and Does Not Involve Supraspinal Sites

Drugs that enhance GABAergic inhibition alleviate inflammatory and neuropathic pain after spinal application. This antihyperalgesia occurs mainly through GABAA receptors (GABAARs) containing α2 subunits (α2-GABAARs). Previous work indicates that potentiation of these receptors in the spinal cord evokes profound antihyperalgesia also after systemic administration, but possible synergistic or antagonistic actions of supraspinal α2-GABAARs on spinal antihyperalgesia have not yet been addressed. Here we generated two lines of GABAAR-mutated mice, which either lack α2-GABAARs specifically from the spinal cord, or, which express only benzodiazepine-insensitive α2-GABAARs at this site. We analyzed the consequences of these mutations for antihyperalgesia evoked by systemic treatment with the novel non-sedative benzodiazepine site agonist HZ166 in neuropathic and inflammatory pain. Wild-type mice and both types of mutated mice had similar baseline nociceptive sensitivities and developed similar hyperalgesia. However, antihyperalgesia by systemic HZ166 was reduced in both mutated mouse lines by about 60% and was virtually indistinguishable from that of global point-mutated mice, in which all α2-GABAARs were benzodiazepine insensitive. The major (α2-dependent) component of GABAAR-mediated antihyperalgesia was therefore exclusively of spinal origin, whereas supraspinal α2-GABAARs had neither synergistic nor antagonistic effects on antihyperalgesia. Our results thus indicate that drugs that specifically target α2-GABAARs exert their antihyperalgesic effect through enhanced spinal nociceptive control. Such drugs may therefore be well-suited for the systemic treatment of different chronic pain conditions

Circuit dissection of the role of somatostatin in itch and pain

Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord; however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, particularly the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb+ neurons, and we demonstrate that Nppb+somatostatin+ cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR+ neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch and characterize a contrasting antinociceptive role for the peptide