The role of tonic glycinergic conductance in cerebellar granule cell signalling and the effect of gain‐of‐function mutation

Abstract

Key pointsr A T258F mutation of the glycine receptor increases the receptor affinity to endogenous agonists, modifies single-channel conductance and shapes response decay kinetics.r Glycine receptors of cerebellar granule cells play their functional role not continuously, but when the granule cell layer starts receiving a high amount of excitatory inputs.r Despite their relative scarcity, tonically active glycine receptors of cerebellar granule cells make a significant impact on action potential generation and inter-neuronal crosstalk, and modulate synaptic plasticity in neural networks; extracellular glycine increases probability of postsynaptic response occurrence acting at NMDA receptors and decreases this probability acting at glycine receptors.r Tonic conductance through glycine receptors of cerebellar granule cells is a yet undiscovered element of the biphasic mechanism that regulates processing of sensory inputs in the cerebellum.r A T258F point mutation disrupts this biphasic mechanism, thus illustrating the possible role of the gain-of-function mutations of the glycine receptor in development of neural pathologies.Abstract Functional glycine receptors (GlyRs) have been repeatedly detected in cerebellar granule cells (CGCs), where they deliver exclusively tonic inhibitory signals. The functional role of this signalling, however, remains unclear. Apart from that, there is accumulating evidence of the important role of GlyRs in cerebellar structures in development of neural pathologies such as hyperekplexia, which can be triggered by GlyR gain-of-function mutations. In this research we initially tested functional properties of GlyRs, carrying the yet understudied T258F gain-of-function mutation, and found that this mutation makes significant modifications in GlyR response to endogenous agonists. Next, we clarified the role of tonic GlyR conductance in Following a BSc in genetics and cell biology and Masters in genetic engineering and novel therapeutics, Catherine McLaughlin gained a research position in Seth Grant's laboratory focused on genome editing with primary and differentiated cell screening to develop improved cell assay platforms. Now based in The Roslin Institute, as part of the Gene Therapy Group, her research is concentrated on emerging genetic therapies and their optimization for human clinical trials. C. McLaughlin and others J Physiol 597.9neuronal signalling generated by single CGCs and by neural networks in cell cultures and in living cerebellar tissue of C57Bl-6J mice. We found that GlyRs of CGCs deliver a significant amount of tonic inhibition not continuously, but when the cerebellar granule layer starts receiving substantial excitatory input. Under these conditions tonically active GlyRs become a part of neural signalling machinery allowing generation of action potential (AP) bursts of limited length in response to sensory-evoked signals. GlyRs of CGCs support a biphasic modulatory mechanism which enhances AP firing when excitatory input intensity is low, but suppresses it when excitato...

Multi-target action of β-alanine protects cerebellar tissue from ischemic damage

“…5A, B ). This resembles sensory inputs to CGCs in vivo [ 41 , 47 ]. Then, we recorded APs in response to burst stimulation (5 stimuli applied at 25 Hz, in 30-s intervals between trials).…”

“…To reproduce sensory inputs in CGCs, we implemented an experimental protocol as in our earlier work [ 47 ]. In particular, individual CGCs were recorded for evoked APs in response to stimulation of a single MF in the cerebellar white matter (see Methods for details).…”

“…Restored by β-Ala application, the patterns of APs in CGCs (Fig. 5 ), which resemble sensory signaling in vivo [ 41 , 47 ], suggest a neuroprotective effect regarding the cerebellar sensorimotor function prone to ischemic damage.…”

“…To stimulate a single fiber, we used a θ-glass electrode pulled to ~5 μm tip diameter, filled with a perfusion solution, with wires in both channels connected to the stimulus isolator. After a CGC was patched, the position of the stimulating electrode was probed until excitatory input was evoked in a patched cell [ 47 ]. The intracellular pipette solution for voltage-clamp recordings contained (mM) 117.5 Cs-gluconate, 17.5 CsCl, 10 KOH-HEPES, 10 BAPTA, 8 NaCl, 5 QX-314, 2 Mg-ATP, 0.3 GTP; for current-clamp recordings: 126 K-gluconate, 4 NaCl, 5 HEPES, 15 glucose, 1 MgSO 4 ·7H 2 O, 2 BAPTA, 3 Mg-ATP (295 – 310 mOsm, pH = 7.2); pipette resistance was 7 – 9 MΩ.…”

“…CGCs, which play the main regulatory role in this circuit, are also an established subject of studies on neural cell apoptosis, necrosis and survival [ 46 ], making these cells a suitable focus of ischemia research. In our recent work, we showed that the sensory signal handling by CGCs and PCs demonstrated in vivo [ 41 ] can be reproduced in brain slices [ 47 ], which are highly sensitive to ischemic damage [ 48 , 49 ]. In this context, shifts in excitation-inhibition balance (including that in the cerebellar CGC-PC circuit) are among the most important triggers of ischemia-induced functional disorders, excitotoxicity, and cell death [ 11 , 50 , 51 ].…”