Examining Hippocampal Mossy Fiber Synapses by 3D Electron Microscopy in Wildtype and Kirrel3 Knockout Mice

Abstract

Neural circuits balance excitatory and inhibitory activity and disruptions in this balance are commonly found in neurodevelopmental disorders. Mice lacking the intellectual disability and autism-associated gene Kirrel3 have an excitation-inhibition imbalance in the hippocampus but the precise synaptic changes underlying this functional defect are unknown. Kirrel3 is a homophilic adhesion molecule expressed in dentate gyrus (DG) and GABA neurons. It was suggested that the excitation-inhibition imbalance of hippocampal neurons in Kirrel3 knockout mice is due to loss of mossy fiber (MF) filopodia, which are DG axon protrusions thought to excite GABA neurons and thereby provide feed-forward inhibition to CA3 pyramidal neurons. Fewer filopodial structures were observed in Kirrel3 knockout mice but neither filopodial synapses nor DG en passant synapses, which also excite GABA neurons, were examined. Here, we used serial block-face scanning electron microscopy with 3D reconstruction to define the precise connectivity of MF filopodia and elucidate synaptic changes induced by Kirrel3 loss. Surprisingly, we discovered wildtype MF filopodia do not synapse exclusively onto GABA neurons as previously thought, but instead synapse with similar frequency onto GABA neurons and CA3 neurons. Moreover, Kirrel3 loss selectively reduces MF filopodial synapses onto GABA neurons but not those made onto CA3 neurons or en passant synapses. In sum, the selective loss of MF filopodial synapses with GABA neurons likely underlies the hippocampal activity imbalance observed in Kirrel3 knockout mice and may impact neural function in patients with Kirrel3-dependent neurodevelopmental disorders.

Significance Statement Point mutations and deletions in the gene Kirrel3 are associated with neurodevelopmental disorders including autism, intellectual disability and Jacobsen's syndrome, a chromosomal disorder that frequently includes epilepsy, autism, and intellectual disability. We studied the effect of losing Kirrel3 on synaptic connections in the mouse hippocampus, a brain region critical for learning and memory. We find that not only are a specific subset of synapses missing in Kirrel3 knockouts, but we also discovered a new synaptic connection within the hippocampus. The synaptic changes we found in mice lacking Kirrel3 shed new light on how a defective Kirrel3 gene could cause neurodevelopmental disorders in humans.