Biology subjects

Specht, C. G.

Publications and source records attributed to Specht, C. G..

4 recordsLinked to original sources

bioRxiv2021

Identification of a stereotypic molecular arrangement of glycine receptors at native spinal cord synapses

Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in spinal cord tissue using quantitative super-resolution correlative light and electron microscopy (SR-CLEM). We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant packing densities of about 2000 GlyRs m-2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

molecular biology↗

bioRxiv2021

Sylites: Multipurpose markers for the visualization of inhibitory synapses

We introduce Sylites - small and versatile fluorogenic affinity probes for high-contrast visualization of inhibitory synapses. Having stoichiometric labeling and exceptional selectivity for neuronal gephyrin, a hallmark protein of the inhibitory post-synapse, Sylites enable superior synapse staining compared with antibodies. Combined with super-resolution microscopy, Sylites allow precise nanoscopic measurements of the synapse. In brain tissue, Sylites reveal the three-dimensional distribution of inhibitory synapses within just an hour.

neuroscience↗

bioRxiv2020

Reciprocal stabilisation of glycine receptors and gephyrin scaffold proteins at inhibitory synapses

Postsynaptic scaffold proteins immobilise neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses where they form receptor-scaffold complexes. While several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterise the GlyR and gephyrin dynamics at inhibitory synapses we performed fluorescence time-lapse imaging after photoconversion in order to directly visualise the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilisation of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the crosslinking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared to control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR crosslinking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilisation of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organisation. Statement of significanceThe efficiency of signal transmission between neurons depends strongly on the number of available neurotransmitter receptors in the postsynaptic membrane. Postsynaptic scaffold proteins provide binding sites for receptors, thus setting the gain of synaptic transmission. However, the importance of receptor-scaffold interactions for the stability of the postsynaptic scaffold itself has received relatively little attention. Using time-lapse imaging of glycine receptors and gephyrin scaffolds at inhibitory synapses in spinal cord neurons together with biophysical modelling, we show that receptor mobility controls the exchange but not the total number of gephyrin molecules at the synapse, and predict that glycine receptors and gephyrin scaffolds dynamically organise into different subpopulations with varying degrees of reciprocal stabilisation.

neuroscience↗

bioRxiv2020

Differential homeostatic regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses

Super-resolution imaging of synapses has revealed that key synaptic proteins are dynamically organized within sub-synaptic domains (SSDs). At mixed inhibitory synapses in spinal cord neurons, both GlyRs and GABAARs reside at the same post-synaptic density (PSD). To examine how the different inhibitory receptors are organized and regulated, we carried out dual-color direct stochastic optical reconstruction microscopy (dSTORM). We found that endogenous GlyRs and GABAARs as well as their common scaffold protein gephyrin form SSDs that align with pre-synaptic RIM1/2, thus forming trans-synaptic nanocolumns. Strikingly, GlyRs and GABAARs occupy different sub-synaptic spaces, exhibiting only a partial overlap at mixed inhibitory synapses. When network activity was increased by pharmacological treatment using the K+ channel blocker 4-aminopyridine (4-AP), the GABAAR copy numbers of as well as the number of GABAAR SSDs were reduced, while GlyRs remained largely unchanged. This differential regulation is likely the result of changes in gephyrin phosphorylation that preferentially occurred outside of the SSDs. The total gephyrin content was not altered by 4-AP application. The activity-dependent regulation of GABAARs versus GlyRs suggests that different signaling pathways control their respective sub-synaptic organization. Whereas gephyrin serves as a scaffold protein that upholds GlyR numbers at SSDs, it may act as a switch regulating GABAARs via its phosphorylation state. Taken together, our data reinforce the notion that the precise sub-synaptic organization of GlyRs, GABAARs and gephyrin has functional consequences for the homeostatic regulation of mixed inhibitory synapses. HighlightsAlignment of sub-synaptic domains (SSDs) in trans-synaptic nanocolumns at inhibitory synapses Differential spatial organization of SSDs formed by GlyRs and GABAARs at mixed inhibitory synapses Activity-dependent regulation of GABAARs but not GlyRs at mixed inhibitory synapses Gephyrin phosphorylation is compartmentalized in SSDs within the synaptic scaffold

neuroscience↗