The rates of opioid overdose in the United States quadrupled between 1999 and 2017, reaching a staggering 130 deaths per day. This health epidemic demands innovative solutions that require uncovering the key brain areas and cell types mediating the cause of overdose— opioid-induced respiratory depression. Here, we identify two primary changes to murine breathing after administering opioids. These changes implicate the brainstem’s breathing circuitry which we confirm by locally eliminating the µ-Opioid receptor. We find the critical brain site is the preBötzinger Complex, where the breathing rhythm originates, and use genetic tools to reveal that just 70–140 neurons in this region are responsible for its sensitivity to opioids. Future characterization of these neurons may lead to novel therapies that prevent respiratory depression while sparing analgesia.

Highlights d Synaptic inputs onto mPFC L2/3 pyramidal neurons are reduced in Cntnap2 KO mice d The frequency and amplitude of mEPSCs are reduced in the mPFC of Cntnap2 KO neurons d Decreased density of dendritic excitatory and inhibitory synapses in Cntnap2 KO mice d Phase-modulated spiking to slow LFP oscillations is altered in Cntnap2 KO units

Loss of function mutations in CNTNAP2 cause a syndromic form of autism spectrum disorder (ASD) in humans and produce social deficits, repetitive behaviors, and seizures in mice. Yet, the functional effects of these mutations at the cellular and circuit level remain elusive. Using laser scanning photostimulation, whole-cell recordings, and electron microscopy, we found a dramatic decrease in functional excitatory and inhibitory synaptic inputs in L2/3 medial prefrontal cortex (mPFC) of Cntnap2 knock-out (KO) mice. In accordance with decreased synaptic input, KO mice displayed reduced spine and synapse densities, despite normal intrinsic excitability and dendritic complexity. To determine how this decrease in synaptic inputs alters coordination of neuronal firing patterns in vivo, we recorded mPFC local field potentials (LFP) and unit spiking in head-fixed mice during locomotion and rest. In KO mice, LFP power was not significantly altered at all tested frequencies, but inhibitory neurons showed delayed phase-firing and reduced phase-locking to delta and theta oscillations during locomotion. Excitatory neurons showed similar changes but only to delta oscillations. These findings suggest that profound ASD-related alterations in synaptic inputs can yield perturbed temporal coordination of cortical ensembles.All rights reserved. No reuse allowed without permission.

Opioids are perhaps the most effective analgesics in medicine. However, between 1999 to 2018, over 400,000 people in the United States died from opioid overdose. Excessive opioids make breathing lethally slow and shallow, a side-effect called opioid induced respiratory depression. This doubled-edged sword has sparked the desire to develop novel therapeutics that provide opioid-like analgesia without depressing breathing. One such approach has been the design of so-called 'biased agonists' that signal through some, but not all pathways downstream of the µ-opioid receptor (MOR), the target of morphine and other opioid analgesics. This rationale stems from a study suggesting that MOR-induced ß2-arrestin dependent signaling is responsible for opioid respiratory depression, whereas adenylyl cyclase inhibition produces analgesia. To verify this important result that motivated the 'biased agonist' approach, we re-examined breathing in ß2-arrestin deficient mice and instead find no connection between ß2-arrestin and opioid respiratory depression. This result suggests that any attenuated effect of 'biased agonists' on breathing is through an as-yet defined mechanism.

The rates of opioid overdose in the United States quadrupled between 1999 and 2017, reaching a staggering 130 deaths per day. This health epidemic demands innovative solutions that require uncovering the key brain areas and cell types mediating the cause of overdose—opioid respiratory depression. Here, we identify two primary changes to breathing after administering opioids. These changes implicate the brainstem’s breathing circuitry which we confirm by locally eliminating the μ-Opiate receptor. We find the critical brain site is the origin of the breathing rhythm, the preBötzinger Complex, and use genetic tools to reveal that just 70-140 neurons in this region are responsible for its sensitivity to opioids. Future characterization of these neurons may lead to novel therapies that prevent respiratory depression while sparing analgesia.