DAPK1 binding to GluN2B was prominently reported to mediate ischemic cell death in vivo. DAPK1 and CaMKII bind to the same GluN2B region, and their binding is mutually exclusive. Here, we show that mutating the binding region on GluN2B (L1298A/R1300Q) protected against neuronal cell death induced by cardiac arrest followed by resuscitation. Importantly, the GluN2B mutation selectively abolished only CaMKII, but not DAPK1, binding. During ischemic or excitotoxic insults, CaMKII further accumulated at excitatory synapses, and this accumulation was mediated by GluN2B binding. Interestingly, extra-synaptic GluN2B decreased after ischemia, but its relative association with DAPK1 increased. Thus, ischemic neuronal death requires CaMKII binding to synaptic GluN2B, whereas any potential role for DAPK1 binding is restricted to a different, likely extra-synaptic population of GluN2B.

Here, we use optogenetics and chemogenetics to investigate the contribution of the paraventricular thalamus (PVT) to nucleus accumbens (NAc) pathway in aversion and heroin relapse in two different heroin self-administration models in rats. In one model, rats undergo forced abstinence in the home cage prior to relapse testing, and in the other, they undergo extinction training, a procedure that is likened to cognitive behavioral therapy. We find that the PVT→NAc pathway is both sufficient and necessary to drive aversion and heroin seeking after abstinence, but not extinction. The ability of extinction to reduce this pathway's contribution to heroin relapse is accompanied by a loss of synaptic plasticity in PVT inputs onto a specific subset of NAc neurons. Thus, extinction may exert therapeutic reductions in opioid seeking by altering synaptic plasticity within the PVT→NAc pathway, resulting in reduced aversion during opioid withdrawal as well as reduced relapse propensity.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling, but its role in pathological glutamate signaling (excitotoxicity) remains less clear, with indications for both neuro-toxic and neuro-protective functions. Here, the role of CaMKII in ischemic injury is assessed utilizing our mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). CaMKII inhibition (with tatCN21 or tatCN19o) at clinically relevant time points (30 min after resuscitation) greatly reduces neuronal injury. Importantly, CaMKII inhibition also works in combination with mild hypothermia, the current standard of care. The relevant drug target is specifically Ca2+-independent "autonomous" CaMKII activity generated by T286 autophosphorylation, as indicated by substantial reduction in injury in autonomy-incompetent T286A mutant mice. In addition to reducing cell death, tatCN19o also protects the surviving neurons from functional plasticity impairments and prevents behavioral learning deficits, even at extremely low doses (0.01 mg/kg), further highlighting the clinical potential of our findings.

Projections from the nucleus accumbens to the ventral pallidum (VP) regulate relapse in animal models of addiction. The VP contains GABAergic (VPGABA) and glutamatergic (VPGlu) neurons, and a subpopulation of GABAergic neurons co-express enkephalin (VPPenk). Rabies tracing reveals that VPGlu and VPPenk neurons receive preferential innervation from upstream D1- relative to D2-expressing accumbens neurons. Chemogenetic stimulation of VPGlu neurons inhibits, whereas stimulation of VPGABA and VPPenk neurons potentiates cocaine seeking in mice withdrawn from intravenous cocaine self-administration. Calcium imaging reveals cell type-specific activity patterns when animals learn to suppress drug seeking during extinction training versus engaging in cue-induced cocaine seeking. During cued seeking, VPGABA neurons increase their overall activity, and VPPenk neurons are selectively activated around nose pokes for cocaine. In contrast, VPGlu neurons increase their spike rate following extinction training. These data show that VP subpopulations differentially encode and regulate cocaine seeking, with VPPenk and VPGABA neurons facilitating and VPGlu neurons inhibiting cocaine seeking.

GABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABAARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABAAR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.