Local externalization of phosphatidylserine mediates developmental synaptic pruning by microglia.

Neuronal circuit assembly requires the fine balance between synapse formation and elimination. Microglia, through the elimination of supernumerary synapses, have an established role in this process. While the microglial receptor TREM2 and the soluble complement proteins C1q and C3 are recognized as key players, the neuronal molecular components that specify synapses to be eliminated are still undefined. Here, we show that exposed phosphatidylserine (PS) represents a neuronal "eat-me" signal involved in microglial-mediated pruning. In hippocampal neuron and microglia co-cultures, synapse elimination can be partially prevented by blocking accessibility of exposed PS using Annexin V or through microglial loss of TREM2. In vivo, PS exposure at both hippocampal and retinogeniculate synapses and engulfment of PS-labeled material by microglia occurs during established developmental periods of microglial-mediated synapse elimination. Mice deficient in C1q, which fail to properly refine retinogeniculate connections, have elevated presynaptic PS exposure and reduced PS engulfment by microglia. These data provide mechanistic insight into microglial-mediated synapse pruning and identify a novel role of developmentally regulated neuronal PS exposure that is common among developing brain structures.

CSMD1 regulates brain complement activity and circuit development.

Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple Domains 1 (CSMD1) can regulate complement activity in vitro, is expressed in the brain, and is associated with increased schizophrenia risk. Beyond this, little is known about CSMD1 including whether it regulates complement activity in the brain or otherwise plays a role in neurodevelopment. We used biochemical, immunohistochemical, and proteomic techniques to examine the regional, cellular, and subcellular distribution as well as protein interactions of CSMD1 in the brain. To evaluate whether CSMD1 is involved in complement-mediated synapse elimination, we examined Csmd1-knockout mice and CSMD1-knockout human stem cell-derived neurons. We interrogated synapse and circuit development of the mouse visual thalamus, a process that involves complement pathway activity. We also quantified complement deposition on synapses in mouse visual thalamus and on cultured human neurons. Finally, we assessed uptake of synaptosomes by cultured microglia. We found that CSMD1 is present at synapses and interacts with complement proteins in the brain. Mice lacking Csmd1 displayed increased levels of complement component C3, an increased colocalization of C3 with presynaptic terminals, fewer retinogeniculate synapses, and aberrant segregation of eye-specific retinal inputs to the visual thalamus during the critical period of complement-dependent refinement of this circuit. Loss of CSMD1 in vivo enhanced synaptosome engulfment by microglia in vitro, and this effect was dependent on activity of the microglial complement receptor, CR3. Finally, human stem cell-derived neurons lacking CSMD1 were more vulnerable to complement deposition. These data suggest that CSMD1 can function as a regulator of complement-mediated synapse elimination in the brain during development.

Perirhinal cortex learns a predictive map of the task environment.

Goal-directed tasks involve acquiring an internal model, known as a predictive map, of relevant stimuli and associated outcomes to guide behavior. Here, we identified neural signatures of a predictive map of task behavior in perirhinal cortex (Prh). Mice learned to perform a tactile working memory task by classifying sequential whisker stimuli over multiple training stages. Chronic two-photon calcium imaging, population analysis, and computational modeling revealed that Prh encodes stimulus features as sensory prediction errors. Prh forms stable stimulus-outcome associations that can progressively be decoded earlier in the trial as training advances and that generalize as animals learn new contingencies. Stimulus-outcome associations are linked to prospective network activity encoding possible expected outcomes. This link is mediated by cholinergic signaling to guide task performance, demonstrated by acetylcholine imaging and systemic pharmacological perturbation. We propose that Prh combines error-driven and map-like properties to acquire a predictive map of learned task behavior.

PERIRHINAL CORTEX LEARNS A PREDICTIVE MAP (INTERNAL MODEL) OF THE TASK ENVIRONMENT.

Goal-directed tasks involve acquiring an internal model, known as a predictive map, of relevant stimuli and associated outcomes to guide behavior. Here, we identified neural signatures of a predictive map of task behavior in perirhinal cortex (Prh). Mice learned to perform a tactile working memory task by classifying sequential whisker stimuli over multiple training stages. Chemogenetic inactivation demonstrated that Prh is involved in task learning. Chronic two-photon calcium imaging, population analysis, and computational modeling revealed that Prh encodes stimulus features as sensory prediction errors. Prh forms stable stimulus-outcome associations that expand in a retrospective manner and generalize as animals learn new contingencies. Stimulus-outcome associations are linked to prospective network activity encoding possible expected outcomes. This link is mediated by cholinergic signaling to guide task performance, demonstrated by acetylcholine imaging and perturbation. We propose that Prh combines error-driven and map-like properties to acquire a predictive map of learned task behavior.