THE HONEYCOMB SYNAPSE: A STORY OF SYNAPTIC DIVERSITY AND VULNERABILITY

The diversity of synapses between neurons is critical for the specialization of neural circuits that underlie a broad range of behaviours. One of the simplest neuronal circuits in the mammalian nervous system is the spinal reflex arc, comprising a direct connection between sensory and motor neurons that facilitates proprioceptive feedback. Given its simplicity, one might expect this circuit to rely on a relatively homogeneous population of rudimentary synapses. To investigate this, we used transgenic fluorescent reporter lines, immunohistochemistry, super-resolution fluorescence microscopy, and machine learning-based image analysis. This revealed unexpected synaptic complexity within the reflex arc. We identified a novel subtype of Ia afferent synapse, distinguished by its large size (1–5 µm) and complex morphology, including multiple perforations within the postsynaptic density (PSD), opposed to large VGLUT1-expressing presynaptic boutons. Super-resolution imaging revealed a striking architecture—5–6 molecular scaffolding clusters arranged around each perforation—prompting us to define these structures as Honeycomb Synapses. While the PSD facilitates glutamatergic transmission, the perforations contain gap junction proteins for electrical signalling. Using a novel multiplexed microscopy approach, we investigated the vulnerability of synapse subtypes in an inducible model of sporadic Amyotrophic Lateral Sclerosis (sALS). While overall VGLUT1 synapses were reduced in number by ~20%, Honeycomb Synapses were entirely lost, indicating a highly selective vulnerability. This study highlights the importance of synaptic diversity within even the simplest circuits. The unique architecture of Honeycomb Synapses may enable mixed electrical chemical signalling, and their selective loss suggests a critical role in motor circuit stability.