0044 Sleep Need-dependent Plasticity of a Thalamic Circuit Generates Persistence of Sleep Drive

Abstract Introduction Prolonged wakefulness leads to persistent, deep recovery sleep. However, the neuronal circuits mediating this process remain elusive. Here, starting from a large circuit screen, we identify and characterize a group of thalamic nucleus reuniens (RE) neurons activated by sleep pressure and required for sleep homeostasis. Methods To identify upstream excitatory neuronal populations inducing NREM sleep, we performed rabies virus-mediated screening in mice. Sleep/wake states were assayed using tethered EEG/EMG recordings. Activation or inhibition of neuronal activity was performed using chemogenetic and/or optogenetic techniques. Behavior during optogenetic RE activation was assessed using video recordings. To genetically access the sleep pressure-activated RE neurons, Targeted Recombination in Active Populations (TRAP2) mice were used. Viral expression of caspase3 was used for selective ablation of cells. For projection analyses, virally expressed Synaptophysin was used as a presynaptic marker. To visually examine synaptic connectivity, we performed enhanced Green fluorescent protein Reconstruction Across Synaptic Partners (eGRASP) analysis. Functional connectivity between RE to downstream neurons was tested by slice patch-clamp recordings. Results From a circuit screen in mice, we identified a group of excitatory thalamic RE neurons activated by sleep pressure and required for sleep homeostasis. Optogenetic activation of these neurons leads to an unusual phenotype: sleep-preparatory behaviors (e.g., nesting) followed by prolonged, intense sleep resembling recovery sleep. The activity of RE neurons (assessed by Fos) is increased by sleep deprivation (SD), and ablation of these Fos+ RE cells markedly impairs recovery sleep following SD. Moreover, inhibiting RE activity during SD impairs subsequent recovery sleep, suggesting that these neurons signal sleep need. RE neurons act upstream of sleep-promoting zona incerta (ZI) cells, and, remarkably, SD triggers plasticity of this circuit to strengthen its connectivity, as assessed by eGRASP analysis and patch-clamp recordings. Finally, our results indicate that CaMKII signaling is required for morphological plasticity of the RE-ZI circuit and persistence of homeostatic NREM sleep. Conclusion A subset of RE neurons encode sleep pressure and generate persistent NREM sleep, via a projection to sleep-promoting ZI neurons. Increased sleep need triggers plasticity of the RE-ZI projection which enhances functional connectivity facilitating the transmission of homeostatic sleep drive. Support (if any) NIH grants: R01NS094571, R35NS122181, S10OD023548, NS05027