Glycine Receptors in Spinal Nociceptive Control-An Update

Diminished inhibitory control of spinal nociception is one of the major culprits of chronic pain states. Restoring proper synaptic inhibition is a well-established rational therapeutic approach explored by several pharmaceutical companies. A particular challenge arises from the need for site-specific intervention to avoid deleterious side effects such as sedation, addiction, or impaired motor control, which would arise from wide-range facilitation of inhibition. Specific targeting of glycinergic inhibition, which dominates in the spinal cord and parts of the hindbrain, may help reduce these side effects. Selective targeting of the α3 subtype of glycine receptors (GlyRs), which is highly enriched in the superficial layers of the spinal dorsal horn, a key site of nociceptive processing, may help to further narrow down pharmacological intervention on the nociceptive system and increase tolerability. This review provides an update on the physiological properties and functions of α3 subtype GlyRs and on the present state of related drug discovery programs

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

Nociception in the glycine receptor deficient mutant mouse spastic

Glycine receptors (GlyRs) are the primary mediators of fast inhibitory transmission in the mammalian spinal cord, where they modulate sensory and motor signaling. Mutations in GlyR genes as well as some other genes underlie the hereditary disorder hyperekplexia, characterized by episodic muscle stiffness and exaggerated startle responses. Here, we have investigated pain-related behavior and GlyR expression in the spinal cord of the GlyR deficient mutant mouse spastic (spa). In spastic mice, the GlyR number is reduced due to a β subunit gene (Glrb) mutation resulting in aberrant splicing of GlyRβ transcripts. Via direct physical interaction with the GlyR anchoring protein gephyrin, this subunit is crucially involved in the postsynaptic clustering of heteromeric GlyRs. We show that the mutation differentially affects aspects of the pain-related behavior of homozygous Glrb(spa)/Glrb(spa) mice. While response latencies to noxious heat were unchanged, chemically induced pain-related behavior revealed a reduction of the licking time and an increase in flinching in spastic homozygotes during both phases of the formalin test. Mechanically induced nocifensive behavior was reduced in spastic mice, although hind paw inflammation (by zymosan) resulted in allodynia comparable to wild-type mice. Immunohistochemical staining of the spinal cord revealed a massive reduction of dotted GlyRα subunit immunoreactivity in both ventral and dorsal horns, suggesting a reduction of clustered receptors at synaptic sites. Transcripts for all GlyRα subunit variants, however, were not reduced throughout the dorsal horn of spastic mice. These findings suggest that the loss of functional GlyRβ subunits and hence synaptically localized GlyRs compromises sensory processing differentially, depending on stimulus modality

Neurosteroids Mediate Neuroprotection in an In Vitro Model of Hypoxic/Hypoglycaemic Excitotoxicity via δ-GABAA_{A} Receptors without Affecting Synaptic Plasticity

Neurosteroids and benzodiazepines are modulators of the GABA$_{A}$ receptors, thereby causing anxiolysis. Furthermore, benzodiazepines such as midazolam are known to cause adverse side-effects on cognition upon administration. We previously found that midazolam at nanomolar concentrations (10 nM) blocked long-term potentiation (LTP). Here, we aim to study the effect of neurosteroids and their synthesis using XBD173, which is a synthetic compound that promotes neurosteroidogenesis by binding to the translocator protein 18 kDa (TSPO), since they might provide anxiolytic activity with a favourable side-effect profile. By means of electrophysiological measurements and the use of mice with targeted genetic mutations, we revealed that XBD173, a selective ligand of the translocator protein 18 kDa (TSPO), induced neurosteroidogenesis. In addition, the exogenous application of potentially synthesised neurosteroids (THDOC and allopregnanolone) did not depress hippocampal CA1-LTP, the cellular correlate of learning and memory. This phenomenon was observed at the same concentrations that neurosteroids conferred neuroprotection in a model of ischaemia-induced hippocampal excitotoxicity. In conclusion, our results indicate that TSPO ligands are promising candidates for post-ischaemic recovery exerting neuroprotection, in contrast to midazolam, without detrimental effects on synaptic plasticity

γ1 GABAA_{A}Receptors in Spinal Nociceptive Circuits

GABAergic neurons and GABA$_{A}$receptors (GABA$_{A}$Rs) are critical elements of almost all neuronal circuits. Most GABA$_{A}$Rs of the CNS are heteropentameric ion channels composed of two α, two β, and one γ subunits. These receptors serve as important drug targets for benzodiazepine (BDZ) site agonists, which potentiate the action of GABA at GABA$_{A}$Rs. Most GABA$_{A}$R classifications rely on the heterogeneity of the α subunit (α1–α6) included in the receptor complex. Heterogeneity of the γ subunits (γ1–γ3), which mediate synaptic clustering of GABA$_{A}$Rs and contribute, together with α subunits, to the benzodiazepine (BDZ) binding site, has gained less attention, mainly because γ2 subunits greatly outnumber the other γ subunits in most brain regions. Here, we have investigated a potential role of non-γ2 GABA$_{A}$Rs in neural circuits of the spinal dorsal horn, a key site of nociceptive processing. Female and male mice were studied. We demonstrate that besides γ2 subunits, γ1 subunits are significantly expressed in the spinal dorsal horn, especially in its superficial layers. Unlike global γ2 subunit deletion, which is lethal, spinal cord-specific loss of γ2 subunits was well tolerated. GABA$_{A}$R clustering in the superficial dorsal horn remained largely unaffected and antihyperalgesic actions of HZ-166, a nonsedative BDZ site agonist, were partially retained. Our results thus suggest that the superficial dorsal horn harbors functionally relevant amounts of γ1 subunits that support the synaptic clustering of GABA$_{A}$Rs in this site. They further suggest that γ1 containing GABA$_{A}$Rs contribute to the spinal control of nociceptive information flow

Ionotropic glutamate receptors of amacrine cells of the mouse retina

The mammalian retina contains approximately 30 different morphological types of amacrine cells, receiving glutamatergic input from bipolar cells. In this study, we combined electrophysiological and pharmacological techniques in order to study the glutamate receptors expressed by different types of amacrine cells. Whole-cell currents were recorded from amacrine cells in vertical slices of the mouse retina. During the recordings the cells were filled with Lucifer Yellow/Neurobiotin allowing classification as wide-field or narrow-field amacrine cells. Amacrine cell recordings were also carried out in a transgenic mouse line whose glycinergic amacrine cells express enhanced green fluorescent protein (EGFP). Agonist-induced currents were elicited by exogenous application of NMDA, AMPA, and kainate (KA) while holding cells at −75 mV. Using a variety of specific agonists and antagonists (NBQX, AP5, cyclothiazide, GYKI 52466, GYKI 53655, SYM 2081) responses mediated by AMPA, KA, and NMDA receptors could be dissected. All cells (n = 300) showed prominent responses to non-NMDA agonists. Some cells expressed AMPA receptors exclusively and some cells expressed KA receptors exclusively. In the majority of cells both receptor types could be identified. NMDA receptors were observed in about 75% of the wide-field amacrine cells and in less than half of the narrow-field amacrine cells. Our results confirm that different amacrine cell types express distinct sets of ionotropic glutamate receptors, which may be critical in conferring their unique temporal responses to this diverse neuronal class

Targeting the interaction of GABAB_{B} receptors with CaMKII with an interfering peptide restores receptor expression after cerebral ischemia and inhibits progressive neuronal death in mouse brain cells and slices

Cerebral ischemia is the leading cause for long-term disability and mortality in adults due to massive neuronal death. Currently, there is no pharmacological treatment available to limit progressive neuronal death after stroke. A major mechanism causing ischemia-induced neuronal death is the excessive release of glutamate and the associated overexcitation of neurons (excitotoxicity). Normally, GABA$_{B}$ receptors control neuronal excitability in the brain via prolonged inhibition. However, excitotoxic conditions rapidly downregulate GABA$_{B}$ receptors via a CaMKII-mediated mechanism and thereby diminish adequate inhibition that could counteract neuronal overexcitation and neuronal death. To prevent the deleterious downregulation of GABA$_{B}$ receptors, we developed a cell-penetrating synthetic peptide (R1-Pep) that inhibits the interaction of GABA$_{B}$ receptors with CaMKII. Administration of this peptide to cultured cortical neurons exposed to excitotoxic conditions restored cell surface expression and function of GABA$_{B}$ receptors. R1-Pep did not affect CaMKII expression or activity but prevented its T286 autophosphorylation that renders it autonomously and persistently active. Moreover, R1-Pep counteracted the aberrant downregulation of G protein-coupled inwardly rectifying K$^{+}$ channels and the upregulation of N-type voltage-gated Ca$^{2+}$ channels, the main effectors of GABA$_{B}$ receptors. The restoration of GABA$_{B}$ receptors activated the Akt survival pathway and inhibited excitotoxic neuronal death with a wide time window in cultured neurons. Restoration of GABA$_{B}$ receptors and neuroprotective activity of R1-Pep was verified by using brain slices prepared from mice after middle cerebral artery occlusion (MCAO). Treatment with R1-Pep restored normal GABA$_{B}$ receptor expression and GABA receptor-mediated K$^{+}$ channel currents. This reduced MCAO-induced neuronal excitability and inhibited neuronal death. These results support the hypothesis that restoration of GABA$_{B}$ receptor expression under excitatory conditions provides neuroprotection and might be the basis for the development of a selective intervention to inhibit progressive neuronal death after ischemic stroke

Binary recombinase systems for high-resolution conditional mutagenesis

Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive allele

c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy

Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-Maf$^{EX}$) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-Maf$^{EX}$ neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-Maf$^{EX}$ neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-Maf$^{EX}$ neuron activation. Our study identifies c-Maf$^{EX}$ neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control