Coordinated regulation of CB1 cannabinoid receptors and anandamide metabolism stabilizes network activity during homeostatic scaling down

Neurons express overlapping homeostatic mechanisms to regulate synaptic function and network properties in response to perturbations of neuronal activity. Endocannabinoids (eCBs) are bioactive lipids synthesized in the post-synaptic compartments to regulate synaptic transmission, plasticity, and neuronal excitability primarily through retrograde activation of pre- synaptic cannabinoid receptor type 1 (CB1). The eCB system is well-situated to regulate neuronal network properties and coordinate pre- and post-synaptic activity. However, the role of the eCB system in homeostatic adaptations to neuronal hyperactivity is unknown. To address this issue, we used western blot and targeted lipidomics to measure adaptations in eCB system to bicuculline (BCC)-induced chronic hyperexcitation in mature (>DIV21) cultured rat cortical neurons, and used multielectrode array recording and live-cell imaging of glutamate dynamics to test the effects of pharmacological manipulations of eCB on network activities. We show that BCC-induced chronic hyperexcitation triggers homeostatic downscaling and a coordinated adaptation to enhance tonic eCB signaling. Hyperexcitation triggers first the downregulation of fatty acid amide hydrolase (FAAH), the lipase that degrades the eCB anandamide, then an accumulation of anandamide and related metabolites, and finally a delayed upregulation of surface and total CB1. Additionally, we show that BCC-induced downregulation of surface AMPA-type glutamate receptors (AMPARs) and upregulation of CB1 occur through independent mechanisms. Finally, we show that endocannabinoids support baseline network activities before and after downscaling and is engaged to suppress network activities during adaptation to hyperexcitation. We discuss the implications of our findings in the context of downscaling and homeostatic regulation of oscillatory network activities. Significance statementNeurons are remarkably resilient to perturbations in network activities thanks to the expression of overlapping homeostatic adaptations. In response to network hyperactivity or silencing, neurons respond through regulating excitatory and inhibitory post-synaptic neurotransmitter receptors density, probability of pre-synaptic neurotransmitter release, and/or membrane excitability. The endocannabinoid system is a prominent signaling pathway at many synapses that is known to be involved in multiple forms of short- and long-term synaptic plasticity. Here we find that components of the endocannabinoid system are upregulated in response to chronic hyperexcitation of cultured cortical neurons, and that endocannabinoid signaling is required to maintain network activity but also suppresses network events during hyperexcitation. This work supports a novel tonic homeostatic function for the endocannabinoid system in neurons.