SUMMARYThe 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 neurotoxic 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.

Human subjects. Human samples and accompanying clinical data were collected from ICU patients enrolled in the NIRFS study, a substudy of the MESSI cohort, which is a single-center, prospective cohort of patients admitted to the ICU at the Hospital of the University of Pennsylvania. This study was approved by the IRB of the University of Pennsylvania. Subjects or their available surrogates provided written informed consent.

Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. SK2 channels, expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we show that in mice, hippocampal long-term potentiation (LTP) is impaired as early as 3 hrs after recovery from CA/CPR and that LTP remains impaired for at least 30 days. Treatment with the SK2 channel agonist, 1-EBIO 30 minutes after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post-synaptic mechanisms. Real-time quantitative RT-PCR indicates that CA/CPR does not cause a loss of NMDA receptor mRNA 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels were observed 7 or 30 days after CA/CPR. Further, patch-clamp experiments demonstrate no change in functional synaptic NMDA receptors 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity is reduced for the duration of experiments performed (up to 30 days) and that surprisingly, treatment with 1-EBIO did not prevent CA/CPR-induced loss of synaptic SK channel function. We conclude that CA/CPR causes alterations in post-synaptic signaling that are prevented by treatment with the SK2 agonist 1-EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.

Several studies suggest that voltage-gated calcium currents are involved in generating high frequency burst firing in the subiculum, but the exact nature of these currents remains unknown. Here, we used selective pharmacology, molecular and genetic approaches to implicate Cav3.1-containing T-channels in subicular burst firing, in contrast to several previous reports discounting T-channels as major contributors to subicular neuron physiology. Furthermore, pharmacological antagonism of T-channels, as well as global deletion of CaV3.1 isoform, completely suppressed development of long-term potentiation (LTP) in the CA1-subiculum, but not in the CA3-CA1 pathway. Our results indicate that excitability and synaptic plasticity of subicular neurons relies heavily on T-channels. Hence, T-channels may be a promising new drug target for different cognitive deficits.

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Rationale The mechanisms that mediate age differences during nicotine withdrawal are unclear. Objective This study compared kappa opioid receptor (KOR) activation in naïve and nicotine-treated adolescent and adult rats using behavioral and neurochemical approaches to study withdrawal. Methods The behavioral models used to assess withdrawal included conditioned place and elevated plus maze procedures. Deficits in dopamine transmission in the nucleus accumbens (NAcc) were examined using microdialysis procedures. Lastly, the effects of KOR stimulation and blockade on physical signs produced upon removal of nicotine were examined in adults. Results Nicotine-treated adults displayed a robust aversion to an environment paired with a KOR agonist versus naïve adults. Neither of the adolescent groups displayed a place aversion. KOR activation produced an increase in anxiety-like behavior that was highest in nicotine-treated adults versus all other groups. KOR activation produced a decrease in NAcc dopamine that was largest in nicotine-treated adults versus all other groups. Lastly, KOR activation facilitated physical signs of upon removal of nicotine and KOR blockade reduced this effect. Conclusion Chronic nicotine enhanced the affective, anxiogenic, and neurochemical effects produced by KOR activation in adult rats. Our data suggest that chronic nicotine elicits an increase in KOR function, and this may contribute to nicotine withdrawal since KOR activation facilitated and KOR blockade prevented withdrawal signs upon removal of nicotine. Given that chronic nicotine facilitated the neurochemical effects of KOR agonists in adults but not adolescents, it is suggested that KOR regulation of mesolimbic dopamine may contribute to age differences in nicotine withdrawal.

Objective Sirtuins (Sirt) are a class of deacetylase enzymes that play an important role in cell proliferation. Sirt2 activation produces O-acetylated-ADPribose (OAADPr) which can act as a ligand for transient receptor potential cation channel, M2 (TRPM2). We tested the hypothesis that Sirt2 is activated following global cerebral ischemia and contributes to neuronal injury through activation of TRPM2. Methods Adult male and female mice (8–12 weeks old) C57Bl/6 and TRPM2 knock-out mice were subjected to 8 min of cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). The Sirt2 inhibitor AGK-2 was administered intravenously 30 min after resuscitation. Hippocampal CA1 injury was analyzed at 3 days after CA/CPR. Acute Sirt2 activity was analyzed at 3 and 24 h after CA/CPR. Long-term hippocampal function was assessed using slice electrophysiology 7 days after CA/CPR. Results AGK-2 significantly reduced CA1 injury in WT but not TRPM2 knock-out males and had no effect on CA1 injury in females. Elevated Sirt2 activity was observed in hippocampal tissue from males at 24 h after cardiac arrest and was reduced by AGK-2. In contrast, Sirt2 activity in females was increased at 3 but not 24 h. Finally, we observed long-term benefit of AGK-2 on hippocampal function, with a protection of long-term potentiation at CA1 synapses at 7 and 30 days after ischemia. Conclusions In summary, we observed a male specific activation of Sirt2 that contributes to neuronal injury and functional deficits after ischemia specifically in males. These results are consistent with a role of Sirt2 in activating TRPM2 following global ischemia in a sex specific manner. These results support the growing body of literature showing that oxidative stress mechanisms predominate in males and converge on TRPM2 activation as a mediator of cell death.

Pediatric cardiac arrest (CA) often leads to poor neurologic outcomes, including deficits in learning and memory. The only approved treatment for CA is therapeutic hypothermia, although its utility in the pediatric population remains unclear. This study analyzed the effect of mild therapeutic hypothermia after CA in juvenile mice on hippocampal neuronal injury and the cellular model of learning and memory, termed long-term potentiation (LTP). Juvenile mice were subjected to cardiac arrest and cardiopulmonary resuscitation (CA/CPR) followed by normothermia (37 °C) and hypothermia (30 °C, 32 °C). Histological injury of hippocampal CA1 neurons was performed 3 days after resuscitation using hematoxylin and eosin (H&E) staining. Field excitatory post-synaptic potentials (fEPSPs) were recorded from acute hippocampal slices 7 days after CA/CPR to determine LTP. Synaptic function was impaired 7 days after CA/CPR. Mice exposed to hypothermia showed equivalent neuroprotection, but exhibited sexually dimorphic protection against ischemia-induced impairment of LTP. Hypothermia (32 °C) protects synaptic plasticity more effectively in females, with males requiring a deeper level of hypothermia (30 °C) for equivalent protection. In conclusion, male and female juvenile mice exhibit equivalent neuronal injury following CA/CPR and hypothermia protects both males and females. We made the surprising finding that juvenile mice have a sexually dimorphic response to mild therapeutic hypothermia protection of synaptic function, where males may need a deeper level of hypothermia for equivalent synaptic protection.