Activity in numerous brain regions drives heroin seeking, but no circuits that limit heroin seeking have been identified. Furthermore, the neural circuits controlling opioid choice are unknown. In this study, we examined the role of the infralimbic cortex (IL) to nucleus accumbens shell (NAshell) pathway during heroin choice and relapse. This model yielded subpopulations of heroin versus food preferring rats during choice, and choice was unrelated to subsequent relapse rates to heroin versus food cues, suggesting that choice and relapse are distinct behavioral constructs. Supporting this, inactivation of the IL with muscimol produced differential effects on opioid choice versus relapse. A pathway-specific chemogenetic approach revealed, however, that the IL-NAshell pathway acts as a common limiter of opioid choice and relapse. Furthermore, dendritic spines in IL-NAshell neurons encode distinct aspects of heroin versus food reinforcement. Thus, opioid choice and relapse share a common addiction-limiting circuit in the IL-NAshell pathway.

Hippocampal cell death and cognitive dysfunction are common following global cerebral ischemia across all ages, including children. Most research has focused on preventing neuronal death. Restoration of neuronal function after cell death is an alternative approach (neurorestoration). We previously identified transient receptor potential M2 (TRPM2) ion channels as a potential target for acute neuroprotection and delayed neurorestoration in an adult CA/CPR mouse model. Cardiac arrest/cardiopulmonary resuscitation (CA/CPR) in juvenile (p20-25) mice was used to investigate the role of ion TRPM2 channels in neuroprotection and ischemia-induced synaptic dysfunction in the developing brain. Our novel TRPM2 inhibitor, tatM2NX, did not confer protection against CA1 pyramidal cell death but attenuated synaptic plasticity (long-term plasticity (LTP)) deficits in both sexes. Further, in vivo administration of tatM2NX two weeks after CA/CPR reduced LTP impairments and restored memory function. These data provide evidence that pharmacological synaptic restoration of the surviving hippocampal network can occur independent of neuroprotection via inhibition of TRPM2 channels, providing a novel strategy to improve cognitive recovery in children following cerebral ischemia. Importantly, these data underscore the importance of age-appropriate models in disease research.

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.

Cocaine addiction is a chronic, relapsing disorder characterized by maladaptation in the brain mesolimbic and nigrostriatal dopamine system. Although changes in the properties of D2-receptor-expressing medium spiny neurons (D2-MSNs) and connected striatal circuits following cocaine treatment are known, the contributions of altered D2-receptor (D2R) function in mediating the rewarding properties of cocaine remain unclear. Here, we describe how a 7-day exposure to cocaine alters dopamine signaling by selectively reducing the sensitivity, but not the expression, of nucleus accumbens D2-MSN D2Rs via an alteration in the relative expression and coupling of G protein subunits. This cocaine-induced reduction of D2R sensitivity facilitated the development of the rewarding effects of cocaine as blocking the reduction in G protein expression was sufficient to prevent cocaine-induced behavioral adaptations. These findings identify an initial maladaptive change in sensitivity by which mesolimbic dopamine signals are encoded by D2Rs following cocaine exposure.

Relapse is a two-component process consisting of a highly motivated drug-seeking phase that, if successful, is followed by a drug-using phase resulting in temporary satiation. In rodents, cue-induced drug seeking requires transient synaptic potentiation (t-SP) of cortical glutamatergic synapses on nucleus accumbens core medium spiny neurons, but it is unknown how achieving drug use affects this plasticity. We modeled the two phases of relapse after extinction from cocaine self-administration to assess how cocaine use affects t-SP associated with cue-induced drug seeking.

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.

The dorsal subiculum (dSub) is one of the key structures responsible for the formation of hippocampal memory traces but the contribution of individual ionic currents to its cognitive function is not well studied. Although we recently reported that low-voltage-activated T-type calcium channels (T-channels) are crucial for the burst firing pattern regulation in the dSub pyramidal neurons, their potential role in learning and memory remains unclear. Here we used in vivo local field potential recordings and miniscope calcium imaging in freely behaving mice coupled with pharmacological and genetic tools to address this gap in knowledge. We show that the CaV3.1 isoform of T-channels is critically involved in controlling neuronal activity in the dSub in vivo. Altering neuronal excitability by inhibiting T-channel activity markedly affects calcium dynamics, synaptic plasticity, neuronal oscillations and phase-amplitude coupling in the dSub, thereby disrupting spatial learning. These results provide an important causative link between the CaV3.1 channels, burst firing of dSub neurons and memory formation, thus further supporting the notion that changes in neuronal excitability regulate memory processing. We posit that subicular CaV3.1 T-channels could be a promising novel drug target for cognitive disorders.

A single BDNF microinfusion into prelimbic (PrL) cortex immediately after the last cocaine self-administration session decreases relapse to cocaine-seeking. The BDNF effect is blocked by NMDAR antagonists. To determine whether synaptic activity in putative excitatory projection neurons in PrL cortex is sufficient for BDNF's effect on relapse, the PrL cortex of male rats was infused with an inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) viral vector driven by an αCaMKII promoter. Immediately after the last cocaine self-administration session, rats were injected with clozapine-N-oxide 30 min before an intra-PrL BDNF microinfusion. DREADD-mediated inhibition of the PrL cortex blocked the BDNF-induced decrease in cocaine-seeking after abstinence and cue-induced reinstatement after extinction. Unexpectedly, DREADD inhibition of PrL neurons in PBS-infused rats also reduced cocaine-seeking, suggesting that divergent PrL pathways affect relapse. Next, using a cre-dependent retroviral approach, we tested the ability of DREADD inhibition of PrL projections to the NAc core or the paraventricular thalamic nucleus (PVT) to alter cocaine-seeking in BDNF- and PBS-infused rats. Selective inhibition of the PrL-NAc pathway at the end of cocaine self-administration blocked the BDNF-induced decrease in cocaine-seeking but had no effect in PBS-infused rats. In contrast, selective inhibition of the PrL-PVT pathway in PBS-infused rats decreased cocaine-seeking, and this effect was prevented in BDNF-infused rats. Thus, activity in the PrL-NAc pathway is responsible for the therapeutic effect of BDNF on cocaine-seeking whereas inhibition of activity in the PrL-pPVT pathway elicits a similar therapeutic effect in the absence of BDNF.SIGNIFICANCE STATEMENT The major issue in cocaine addiction is the high rate of relapse. However, the neuronal pathways governing relapse remain unclear. Using a pathway-specific chemogenetic approach, we found that BDNF differentially regulates two key prelimbic pathways to guide long-term relapse. Infusion of BDNF in the prelimbic cortex during early withdrawal from cocaine self-administration decreases relapse that is prevented when neurons projecting from the prelimbic cortex to the nucleus accumbens core are inhibited. In contrast, BDNF restores relapse when neurons projecting from the prelimbic cortex to the posterior paraventricular thalamic nucleus are inhibited. This study demonstrates that two divergent cortical outputs mediate relapse that is regulated in opposite directions by infusing BDNF in the prelimbic cortex during early withdrawal from cocaine.

Ischemic long-term potentiation (iLTP) is a form of synaptic plasticity that occurs in acute brain slices following oxygen-glucose deprivation. In vitro, iLTP can occlude physiological LTP (pLTP) through saturation of plasticity mechanisms. We used our murine cardiac arrest and cardiopulmonary resuscitation (CA/CPR) model to produce global brain ischemia and assess whether iLTP is induced in vivo, contributing to the functionally relevant impairment of pLTP. Adult male mice were subjected to CA/CPR, and slice electrophysiology was performed in the hippocampal CA1 region 7 or 30 days later. We observed increased miniature excitatory postsynaptic current amplitudes, suggesting a potentiation of postsynaptic AMPA receptor function after CA/CPR. We also observed increased phosphorylated GluR1 in the postsynaptic density of hippocampi after CA/CPR. These data support the in vivo induction of ischemia-induced plasticity. Application of a low-frequency stimulus (LFS) to CA1 inputs reduced excitatory postsynaptic potentials in slices from mice subjected to CA/CPR, while having no effects in sham controls. These results are consistent with a reversal, or depotentiation, of iLTP. Further, depotentiation with LFS partially restored induction of pLTP with theta burst stimulation. These data provide evidence for iLTP following in vivo ischemia, which occludes pLTP and likely contributes to network disruptions that underlie memory impairments.

Cerebellar Purkinje cells (PCs) are particularly sensitive to cerebral ischemia, and decreased GABA(A) receptor function following injury is thought to contribute to PC sensitivity to ischemia-induced excitotoxicity. Here we examined the functional properties of the GABA(A) receptors that are spared following ischemia in cultured Purkinje cells from rat and in vivo ischemia in mouse. Using subunit-specific positive modulators of GABA(A) receptors, we observed that oxygen and glucose deprivation (OGD) and cardiac arrest-induced cerebral ischemia cause a decrease in sensitivity to the β(2/3) -subunit-preferring compound, etomidate. However, sensitivity to propofol, a β-subunit-acting compound that modulates β(1-3) -subunits, was not affected by OGD. The α/γ-subunit-acting compounds, diazepam and zolpidem, were also unaffected by OGD. We performed single-cell reverse transcription-polymerase chain reaction on isolated PCs from acutely dissociated cerebellar tissue and observed that PCs expressed the β(1) -subunit, contrary to previous reports examining GABA(A) receptor subunit expression in PCs. GABA(A) receptor β(1) -subunit protein was also detected in cultured PCs by western blot and by immunohistochemistry in the adult mouse cerebellum and levels remained unaffected by ischemia. High concentrations of loreclezole (30 μm) inhibited PC GABA-mediated currents, as previously demonstrated with β(1) -subunit-containing GABA(A) receptors expressed in heterologous systems. From our data we conclude that PCs express the β(1) -subunit and that there is a greater contribution of β(1) -subunit-containing GABA(A) receptors following OGD.

Myelin, the insulating sheath around axons, supports axon function. An important question is the impact of mild myelin disruption. In the absence of the myelin protein proteolipid protein (PLP1), myelin is generated but with age, axonal function/maintenance is disrupted. Axon disruption occurs in Plp1-null mice as early as 2 months in cortical projection neurons. High-volume cellular quantification techniques revealed a region-specific increase in oligodendrocyte density in the olfactory bulb and rostral corpus callosum that increased during adulthood. A distinct proliferative response of progenitor cells was observed in the subventricular zone (SVZ), while the number and proliferation of parenchymal oligodendrocyte progenitor cells was unchanged. This SVZ proliferative response occurred prior to evidence of axonal disruption. Thus, a novel SVZ response contributes to the region-specific increase in oligodendrocytes in Plp1-null mice. Young adult Plp1-null mice exhibited subtle but substantial behavioral alterations, indicative of an early impact of mild myelin disruption.

Kappa opioid receptor (KOR) agonists have known anti-addiction properties and can reduce drug seeking. Their potential for clinical use has largely been daunted by their aversive properties mediated through p38 MAPK signaling. Here we examined the therapeutic potential of the KOR agonist U50,488 (U50) to reduce cocaine seeking in a self-administration model. Following cocaine self-administration and 7 days of forced home-cage abstinence, rats were administered a single dose of U50 (5 mg/kg, i.p.) 30 min prior to the first extinction training session, wherein cocaine and the discrete cocaine-paired cues were no longer available. U50 reduced cocaine seeking on this first extinction session, but did not alter extinction training over subsequent days. 2 weeks after U50 treatment, rats underwent a test of cue-induced reinstatement, and rats that had received U50 reinstated less than controls. Central inhibition of p38 MAPK at the time of U50 administration prevented its long-term therapeutic effect on reinstatement, but not its acute reduction in drug seeking on extinction day 1. The long-term therapeutic effect of U50 required operant extinction during U50 exposure, extended to cocaine-primed reinstatement, and was not mimicked by another aversive drug, lithium chloride (LiCl). These data suggest U50 elicits its long-term anti-relapse effects through a KOR-p38 MAPK-specific aversive counterconditioning of the operant cocaine-seeking response. A single, albeit aversive treatment that is able to reduce relapse long-term warrants further consideration of the therapeutic potential of KOR agonists in the treatment of addiction.

Exposure to sedative/hypnotic and anesthetic drugs, such as ketamine, during the critical period of synaptogenesis, causes profound neurotoxicity in the developing rodent and primate brains and is associated with poor cognitive outcomes later in life. The subiculum is especially vulnerable to acute neurotoxicity after neonatal exposure to sedative/hypnotic and anesthetic drugs. The subiculum acts as a relay center between the hippocampal complex and various cortical and subcortical brain regions and is also an independent generator of gamma oscillations. Gamma oscillations are vital in neuronal synchronization and play a role in learning and memory during wake and sleep. However, there has been little research examining long-term changes in subicular neurophysiology after neonatal exposure to ketamine. Here we explore the lasting effects of neonatal ketamine exposure on sleep macrostructure as well as subicular neuronal oscillations and synaptic plasticity in rats. During the peak of rodent synaptogenesis at postnatal day 7, rat pups were exposed to either 40 mg/kg of ketamine over 12 h or to volume matched saline vehicle. At weaning age, a subset of rats were implanted with a cortical and subicular electroencephalogram electrode, and at postnatal day 31, we performed in vivo experiments that included sleep macrostructure (divided into the wake, non-rapid eye movement, and rapid eye movement sleep) and electroencephalogram power spectra in cortex and subiculum. In a second subset of ketamine exposed animals, we conducted ex vivo studies of long-term potentiation (LTP) experiments in adolescent rats. Overall, we found that neonatal exposure to ketamine increased subicular gamma oscillations during non-rapid eye movement sleep but it did not alter sleep macrostructure. Also, we observed a significant decrease in subicular LTP. Gamma oscillations during non-rapid eye movement sleep are implicated in memory formation and consolidation, while LTP serves as a surrogate for learning and memory. Together these results suggest that lasting functional changes in subiculum circuitry may underlie neurocognitive impairments associated with neonatal exposure to anesthetic agents.

We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage-derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions.

The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a central regulator of learning and memory, which poses a problem for targeting it therapeutically. Indeed, our study supports prior conclusions that long-term interference with CaMKII signaling can erase pre-formed memories. By contrast, short-term pharmacological CaMKII inhibition with tatCN19o interfered with learning in mice only mildly and transiently (for less than 1 h) and did not at all reverse pre-formed memories. This was at ≥500fold of the dose that protected hippocampal neurons from cell death after a highly clinically relevant pig model of transient global cerebral ischemia: ventricular fibrillation followed by advanced life support and electrical defibrillation to induce return of spontaneous circulation. Of additional importance for therapeutic development, cardiovascular safety studies in mice and pig did not indicate any concerns with acute tatCN19o injection. Taken together, even though prolonged interference with CaMKII signaling can erase memory, acute short-term CaMKII inhibition with tatCN19o did not cause such retrograde amnesia that would pose a contraindication for therapy.

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a central regulator of learning and memory, which poses a problem for targeting it therapeutically. Indeed, our study supports prior conclusions that long-term interference with CaMKII signaling can erase pre-formed memories. By contrast, short-term pharmacological CaMKII inhibition with the neuroprotective peptide tatCN19o interfered with learning in mice only mildly and transiently (for less than 1 h) and did not at all reverse pre-formed memories. These results were obtained with ≥500-fold of the dose that protected hippocampal neurons from cell death after a highly clinically relevant pig model of transient global cerebral ischemia: ventricular fibrillation followed by advanced life support and electrical defibrillation to induce the return of spontaneous circulation. Of additional importance for therapy development, our preliminary cardiovascular safety studies in mice and pig did not indicate any concerns with acute tatCN19o injection. Taken together, although prolonged interference with CaMKII signaling can erase memory, acute short-term CaMKII inhibition with tatCN19o did not cause such retrograde amnesia that would pose a contraindication for therapy.

TRPM2 channels have been suggested to play a role in ischemic neuronal injury, specifically in males. A major hindrance to TRPM2 research has been the lack of specific TRPM2 inhibitors. The current study characterized the specificity and neuroprotective efficacy of a novel TRPM2 inhibitor.

Perivascular stromal cells (PSCs) are a recently identified cell type that comprises a small percentage of the platelet derived growth factor receptor-β+ cells within the CNS perivascular space. PSCs are activated following injury to the brain or spinal cord, expand in number and contribute to fibrotic scar formation within the injury site. Beyond fibrosis, their high density in the lesion core makes them a potential significant source of signals that act on neural cells adjacent to the lesion site.

Allopregnanolone (ALLO) is a neurosteroid that has many functions in the brain, most notably neuroprotection and modulation of gamma-amino butyric acid (GABA) neurotransmission. Using a mouse model of cardiac arrest and cardiopulmonary resuscitation, we have previously demonstrated that ALLO protects cerebellar Purkinje cells (PCs) from ischemia in a GABA(A) receptor-dependent manner. In this study we examined the effect of sex on ALLO neuroprotection, observing that low dose ALLO (2 mg/kg) provided greater neuroprotection in females compared to males. At a higher dose of ALLO (8 mg/kg), both sexes were significantly protected from ischemic damage. Using an acute cerebellar slice preparation, whole cell voltage clamp recordings were made from PCs. Spontaneous inhibitory post synaptic currents (IPSCs) were analyzed and the response to physiological ALLO (10 nM) was significantly greater in female PCs compared to male. In contrast, recordings of miniature IPSCs, did not exhibit a sex difference in response to ALLO, suggesting that ALLO affects males and females differentially through a mechanism other than binding postsynaptic GABA(A) receptors. We conclude that the female brain has greater sensitivity to ALLO mediated potentiation of GABAergic neurotransmission, contributing to increased neuroprotection.

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.