6-Sulphated Chondroitins Have a Positive Influence on Axonal Regeneration

Chondroitin sulphate proteoglycans (CSPGs) upregulated in the glial scar inhibit axon regeneration via their sulphated glycosaminoglycans (GAGs). Chondroitin 6-sulphotransferase-1 (C6ST-1) is upregulated after injury leading to an increase in 6-sulphated GAG. In this study, we ask if this increase in 6-sulphated GAG is responsible for the increased inhibition within the glial scar, or whether it represents a partial reversion to the permissive embryonic state dominated by 6-sulphated glycosaminoglycans (GAGs). Using C6ST-1 knockout mice (KO), we studied post-injury changes in chondroitin sulphotransferase (CSST) expression and the effect of chondroitin 6-sulphates on both central and peripheral axon regeneration. After CNS injury, wild-type animals (WT) showed an increase in mRNA for C6ST-1, C6ST-2 and C4ST-1, but KO did not upregulate any CSSTs. After PNS injury, while WT upregulated C6ST-1, KO showed an upregulation of C6ST-2. We examined regeneration of nigrostriatal axons, which demonstrate mild spontaneous axon regeneration in the WT. KO showed many fewer regenerating axons and more axonal retraction than WT. However, in the PNS, repair of the median and ulnar nerves led to similar and normal levels of axon regeneration in both WT and KO. Functional tests on plasticity after the repair also showed no evidence of enhanced plasticity in the KO. Our results suggest that the upregulation of 6-sulphated GAG after injury makes the extracellular matrix more permissive for axon regeneration, and that the balance of different CSs in the microenvironment around the lesion site is an important factor in determining the outcome of nervous system injury

A Unified Model of the GABA(A) Receptor Comprising Agonist and Benzodiazepine Binding Sites

We present a full-length α(1)β(2)γ(2) GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and the backbone of β(2)S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy calculations. Muscimol key interactions are predicted to be α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and β(2)F200. Furthermore, we argue that a water molecule could mediate further interactions between muscimol and the backbone of β(2)S156 and β(2)Y157. DZP is predicted to bind with interactions comparable to those of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines α(1)T206 and γ(2)T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important α(1)H101 and the N-methyl group near α(1)Y159, α(1)T206, and α(1)Y209. We present a binding mode of DZP in which the pending phenyl moiety of DZP is buried in the binding pocket and thus shielded from solvent exposure. Our full length GABA(A) receptor is made available as Model S1

The expression of GABA(A) beta subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells

The subunit composition of GABA(A) receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on α and γ subunits, with little attention given to β subunits. Dentate gyrus granule cells (DGGCs) express all three β subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate ‘phasic’ and ‘tonic’ transmission, respectively. To investigate the subcellular distribution of the β subunits we have utilized the patch-clamp technique to compare the properties of ‘tonic’ and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20–26 wild-type (WT), β(2)(−/−), β(2N265S) (etomidate-insensitive), α(1)(−/−) and δ(−/−) mice. Deletion of either the β(2) or the δ subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the δ subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the β(2/3) subunit-selective agent etomidate was significantly reduced for DGGCs derived from β(2N265S) mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on α(4)(−/−) mice, identify a population of extrasynaptic α(4)β(2)δ receptors, whereas synaptic GABA(A) receptors appear to primarily incorporate the β(3) subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating α(5) and γ(2) subunits. Deletion of the β(2) subunit had no effect on the diazepam-induced current and therefore these extrasynaptic receptors do not contain this subunit. The unambiguous identification of these distinct pools of synaptic and extrasynaptic GABA(A) receptors should aid our understanding of how they act in harmony, to regulate hippocampal signalling in health and disease

Neonatal development of the rat visual cortex: synaptic functions of GABA(A) receptor alpha subunits

Each GAB