Researchers have discovered surprisingly low numbers of glycine receptors (GlyRs) in the hippocampus, a brain region crucial for learning and memory. The findings, published in bioRxiv on , challenge conventional understanding of inhibitory neurotransmission in the brain and open new avenues for investigating neurological disorders.
GlyRs are ligand-gated ion channels responsible for mediating fast inhibitory neurotransmission in the central nervous system. While abundant in the spinal cord and brainstem, their presence and function in the hippocampus have been less clear. Previous studies documented the mRNA transcript for the GlyRβ subunit in various brain areas, but quantifying the actual protein expression proved difficult due to a lack of reliable antibodies.
To overcome this challenge, a team led by Serena Camuso at NeuroBicêtre, Inserm U1195 and Bert Brône at UHasselt, utilized single molecule localization microscopy (SMLM). This highly sensitive technique allowed them to visualize and count individual GlyR complexes at synapses. The researchers employed a genetically modified mouse model expressing mEos4b-tagged GlyRβ subunits, enabling precise identification of the receptors.
The study focused on the molecular layer of the dentate gyrus and the stratum radiatum of the CA3 and CA1 regions of the hippocampus. Analysis revealed a strikingly low density of GlyRs compared to the spinal cord – approximately 3 to 10 detections per synapse in the hippocampus versus nearly 1000 in the spinal cord. This represents a roughly hundredfold difference.
“We observed very few single molecule detections during SMLM, indicating exceedingly low mEos4b-GlyRβ expression in the hippocampus,” the researchers wrote. Further control experiments, including recordings from wildtype mice lacking the mEos4b tag and experiments without UV-induced photoconversion, confirmed the specificity of the signal and ruled out imaging artifacts.
The team estimated that GlyRs are present at only about a quarter of hippocampal synapses, with most synapses containing fewer than 10 receptors. A significant 75% of synapses lacked GlyRs altogether. No substantial differences in receptor copy numbers were observed between the different hippocampal sub-regions examined.
The researchers also addressed the potential for random co-localization of mEos4b detections and gephyrin, a scaffolding protein found at inhibitory synapses. A pixel shift analysis demonstrated that the observed co-localization was not due to chance, further validating the findings.
The study’s methodology involved quantifying the number of mEos4b detections per gephyrin cluster using Icy software. Copy numbers were calculated by dividing detections at synapses by the average number of detections of individual mEos4b-GlyRβ containing receptors, measured across CA3 slices. This approach avoids assumptions about the stoichiometry of GlyR complexes, which remains a topic of debate.
The findings have implications for understanding the role of inhibitory neurotransmission in hippocampal function. The low density of GlyRs suggests that other inhibitory mechanisms, such as GABAergic signaling, may play a more dominant role in regulating neuronal activity in this brain region. Further research is needed to determine the functional consequences of this low GlyR expression and its potential contribution to neurological disorders.
The research also highlights the power of SMLM as a tool for investigating the distribution and abundance of synaptic receptors. The ability to visualize individual receptor complexes provides unprecedented insights into the molecular organization of synapses and opens new possibilities for studying synaptic dysfunction in disease. The study builds on previous work demonstrating the use of SMLM to detect glycine receptors, as reported by researchers in eLife.
The role of the Glycine Receptor β Subunit in synaptic localization is also being investigated, as noted in a recent publication in the Journal of Neuroscience (Google News).
The study’s findings, while preliminary, represent a significant step forward in understanding the complex interplay of inhibitory neurotransmission in the brain and could potentially inform the development of new therapeutic strategies for neurological and psychiatric disorders.
