Brian R. Lee

In Vivo Cocaine Experience Generates Silent Synapses

Studies over the past decade have enunciated silent synapses as prominent cellular substrates for synaptic plasticity in the developing brain. However, little is known about whether silent synapses can be generated postdevelopmentally. Here, we demonstrate that highly salient in vivo experience, such as exposure to cocaine, generates silent synapses in the nucleus accumbens (NAc) shell, a key brain region mediating addiction-related learning and memory. Furthermore, this cocaine-induced generation of silent synapses is mediated by membrane insertions of new, NR2B-containing N-methyl-D-aspartic acid receptors (NMDARs). These results provide evidence that silent synapses can be generated de novo by in vivo experience and thus may act as highly efficient neural substrates for the subsequent experience-dependent synaptic plasticity underlying extremely long-lasting memory.

Bidirectional Modulation of Incubation of Cocaine Craving by Silent Synapse-Based Remodeling of Prefrontal Cortex to Accumbens Projections

Glutamatergic projections from the medial prefrontal cortex (mPFC) to nucleus accumbens (NAc) contribute to cocaine relapse. Here we show that silent synapse-based remodeling of the two major mPFC-to-NAc projections differentially regulated the progressive increase in cue-induced cocaine seeking after withdrawal (incubation of cocaine craving). Specifically, cocaine self-administration in rats generated AMPA receptor-silent glutamatergic synapses within both infralimbic (IL) and prelimbic mPFC (PrL) to NAc projections, measured after 1 day of withdrawal. After 45 days of withdrawal, IL-to-NAc silent synapses became unsilenced/matured by recruiting calcium-permeable (CP) AMPARs, whereas PrL-to-NAc silent synapses matured by recruiting non-CP-AMPARs, resulting in differential remodeling of these projections. Optogenetic reversal of silent synapse-based remodeling of IL-to-NAc and PrL-to-NAc projections potentiated and inhibited, respectively, incubation of cocaine craving on withdrawal day 45. Thus, pro- and antirelapse circuitry remodeling is induced in parallel after cocaine self-administration. These results may provide substrates for utilizing endogenous antirelapse mechanisms to reduce cocaine relapse.

A Silent Synapse-Based Mechanism for Cocaine-Induced Locomotor Sensitization

Locomotor sensitization is a common and robust behavioral alteration in rodents whereby following exposure to abused drugs such as cocaine, the animal becomes significantly more hyperactive in response to an acute drug challenge. Here, we further analyzed the role of cocaine-induced silent synapses in the nucleus accumbens (NAc) shell and their contribution to the development of locomotor sensitization. Using a combination of viral vector-mediated genetic manipulations, biochemistry, and electrophysiology in a locomotor sensitization paradigm with repeated, daily, noncontingent cocaine (15 mg/kg) injections, we show that dominant-negative cAMP-element binding protein (CREB) prevents cocaine-induced generation of silent synapses of young (30 d old) rats, whereas constitutively active CREB is sufficient to increase the number of NR2B-containing NMDA receptors (NMDARs) at synapses and to generate silent synapses. We further show that occupancy of CREB at the NR2B promoter increases and is causally related to the increase in synaptic NR2B levels. Blockade of NR2B-containing NMDARs by administration of the NR2B-selective antagonist Ro256981 directly into the NAc, under conditions that inhibit cocaine-induced silent synapses, prevents the development of cocaine-elicited locomotor sensitization. Our data are consistent with a cellular cascade whereby cocaine-induced activation of CREB promotes CREB-dependent transcription of NR2B and synaptic incorporation of NR2B-containing NMDARs, which generates new silent synapses within the NAc. We propose that cocaine-induced activation of CREB and generation of new silent synapses may serve as key cellular events mediating cocaine-induced locomotor sensitization. These findings provide a novel cellular mechanism that may contribute to cocaine-induced behavioral alterations.

The NMDA Antagonist MK-801 Disrupts Reconsolidation of a Cocaine-Associated Memory for Conditioned Place Preference but Not for Self-Administration in Rats.

Recent research suggests that drug-related memories are reactivated after exposure to environmental cues and may undergo reconsolidation, a process that can strengthen memories. Conversely, reconsolidation may be disrupted by certain pharmacological agents such that the drug-associated memory is weakened. Several studies have demonstrated disruption of memory reconsolidation using a drug-induced conditioned place preference (CPP) task, but no studies have explored whether cocaine-associated memories can be similarly disrupted in cocaine self-administering animals after a cocaine priming injection, which powerfully reinstates drug-seeking behavior. Here we used cocaine-induced CPP and cocaine self-administration to investigate whether the N-methyl-D-aspartate receptor antagonist (+)-5methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) given just prior to reactivation sessions would suppress subsequent cocaine-primed reinstatement (disruption of reconsolidation). Systemic injection of MK-801 (0.05 or 0.20 mg/kg administered intraperitoneally) in rats just prior to reactivation of the cocaine-associated memory in the CPP context attenuated subsequent cocaine-primed reinstatement, while no disruption occurred in rats that did not receive reactivation in the CPP context. However, in rats trained to self-administer cocaine, systemic administration of MK-801 just prior to either of two different types of reactivation sessions had no effect on subsequent cocaine-primed reinstatement of lever-pressing behavior. Thus, systemic administration of MK-801 disrupted the reconsolidation of a cocaine-associated memory for CPP but not for self-administration. These findings suggest that cocaine-CPP and self-administration do not use similar neurochemical processes to disrupt reconsolidation or that cocaine-associated memories in self-administering rats do not undergo reconsolidation, as assessed by lever-pressing behavior under cocaine reinstatement conditions.

Selective presynaptic enhancement of the prefrontal cortex to nucleus accumbens pathway by cocaine.

The nucleus accumbens (NAc) regulates motivated behavior by, in part, processing excitatory synaptic projections from several brain regions. Among these regions, the prefrontal cortex (PFC) and basolateral amygdala, convey executive control and affective states, respectively. Whereas glutamatergic synaptic transmission within the NAc has been recognized as a primary cellular target for cocaine and other drugs of abuse to induce addiction-related pathophysiological motivational states, the understanding has been thus far limited to drug-induced postsynaptic alterations. It remains elusive whether exposure to cocaine or other drugs of abuse influences presynaptic functions of these excitatory projections, and if so, in which projection pathways. Using optogenetic methods combined with biophysical assays, we demonstrate that the presynaptic release probability (Pr) of the PFC-to-NAc synapses was enhanced after short-term withdrawal (1 d) and long-term (45 d) withdrawal from either noncontingent (i.p. injection) or contingent (self-administration) exposure to cocaine. After long-term withdrawal of contingent drug exposure, the Pr was higher compared with i.p. injected rats. In contrast, within the basolateral amygdala afferents, presynaptic Pr was not significantly altered in any of these experimental conditions. Thus, cocaine-induced procedure- and pathway-specific presynaptic enhancement of excitatory synaptic transmission in the NAc. These results, together with previous findings of cocaine-induced postsynaptic enhancement, suggest an increased PFC-to-NAc shell glutamatergic synaptic transmission after withdrawal from exposure to cocaine. This presynaptic alteration may interact with other cocaine-induced cellular adaptations to shift the functional output of NAc neurons, contributing to the addictive emotional and motivational state.

Protection from cyanide-induced brain injury by the Nrf2 transcriptional activator carnosic acid

Cyanide is a life‐threatening, bioterrorist agent, preventing cellular respiration by inhibiting cytochrome c oxidase, resulting in cardiopulmonary failure, hypoxic brain injury, and death within minutes. However, even after treatment with various antidotes to protect cytochrome oxidase, cyanide intoxication in humans can induce a delayed‐onset neurological syndrome that includes symptoms of Parkinsonism. Additional mechanisms are thought to underlie cyanide‐induced neuronal damage, including generation of reactive oxygen species. This may account for the fact that antioxidants prevent some aspects of cyanide‐induced neuronal damage. Here, as a potential preemptive countermeasure against a bioterrorist attack with cyanide, we tested the CNS protective effect of carnosic acid (CA), a pro‐electrophilic compound found in the herb rosemary. CA crosses the blood–brain barrier to up‐regulate endogenous antioxidant enzymes via activation of the Nrf2 transcriptional pathway. We demonstrate that CA exerts neuroprotective effects on cyanide‐induced brain damage in cultured rodent and human‐induced pluripotent stem cell‐derived neurons in vitro, and in vivo in various brain areas of a non‐Swiss albino mouse model of cyanide poisoning that simulates damage observed in the human brain. Cyanide, a potential bioterrorist agent, can produce a chronic delayed‐onset neurological syndrome that includes symptoms of Parkinsonism. Here, cyanide poisoning treated with the proelectrophillic compound carnosic acid, results in reduced neuronal cell death in both in vitro and in vivo models through activation of the Nrf2/ARE transcriptional pathway. Carnosic acid is therefore a potential treatment for the toxic central nervous system (CNS) effects of cyanide poisoning. ARE, antioxidant responsive element; Nrf2 (NFE2L2, Nuclear factor (erythroid‐derived 2)‐like 2).

Searching for Presynaptic NMDA Receptors in the Nucleus Accumbens

The nucleus accumbens shell (NAc) is a key brain region mediating emotional and motivational learning. In rodent models, dynamic alterations have been observed in synaptic NMDA receptors (NMDARs) within the NAc following incentive stimuli, and some of these alterations are critical for acquiring new emotional/motivational states. NMDARs are prominent molecular devices for controlling neural plasticity and memory formation. Although synaptic NMDARs are predominately located postsynaptically, recent evidence suggests that they may also exist at presynaptic terminals and reshape excitatory synaptic transmission by regulating presynaptic glutamate release. However, it remains unknown whether presynaptic NMDARs exist in the NAc and contribute to emotional and motivational learning. In an attempt to identify presynaptically located NMDARs in the NAc, the present study uses slice electrophysiology combined with pharmacological and genetic tools to examine the physiological role of the putative presynaptic NMDARs in rats. Our results show that application of glycine, the glycine-site agonist of NMDARs, potentiated presynaptic release of glutamate at excitatory synapses on NAc neurons, whereas application of 5,7-dichlorokynurenic acid or 7-chlorokynurenic acid, the glycine-site antagonists of NMDARs, produced the opposite effect. However, these seemingly presynaptic NMDAR-mediated effects could not be prevented by application of d-APV, the glutamate-site NMDAR antagonist, and were still present in the mice in which NMDAR NR1 or NR3 subunits were genetically deleted. Thus, rather than suggesting the existence of presynaptic NMDARs, our results support the idea that an unidentified type of glycine-activated substrate may account for the presynaptic effects appearing to be mediated by NMDARs.

Reducing hippocampal cell proliferation in the adult rat does not prevent the acquisition of cocaine-induced conditioned place preference.

Neurogenesis is important for developing certain forms of memory. Recently, hippocampal cell proliferation has been implicated in the development of drug addiction, an extreme form of emotional/motivational pathological memory. Aiming to explore the role of hippocampal neural cell proliferation in cocaine-induced conditioned place preference (CPP), we treated rats with whole brain X-irradiation, which substantially decreases the number of progenitor cells in the subventricular zone of the lateral ventricles and subgranular zone of the dentate gyrus. Surprisingly, there was no difference in the expression of cocaine-induced CPP. These results suggest that the existing neural network, rather than potential new neural circuits mediated by adult neurogenesis, is sufficient for the acquisition of cocaine-induced CPP.

Exposure to Cocaine Regulates Inhibitory Synaptic Transmission in the Nucleus Accumbens

Medium spiny neurons (MSNs) within the nucleus accumbens shell (NAc) function to gate and prioritize emotional/motivational arousals for behavioral output. The neuronal output of NAc MSNs is mainly determined by the integration of membrane excitability and excitatory/inhibitory synaptic inputs. Whereas cocaine-induced alterations at excitatory synapses and membrane excitability have been extensively examined, the overall functional output of NAc MSNs following cocaine exposure is still poorly defined because little is known about whether inhibitory synaptic input to these neurons is affected by cocaine. Here, our results demonstrate multidimensional alterations at inhibitory synapses in NAc neurons following cocaine self-administration in rats. Specifically, the amplitude of miniature IPSCs (mIPSCs) was decreased after 21 d withdrawal from 5 d cocaine self-administration. Upon re-exposure to cocaine after 21 d withdrawal, whereas the amplitude of mIPSCs remained downregulated, the frequency became significantly higher. Furthermore, the reversal potential of IPSCs, which was not significantly altered during withdrawal, became more hyperpolarized upon cocaine re-exposure. Moreover, the relative weight of excitatory and inhibitory inputs to NAc MSNs was significantly decreased after 1 d cocaine withdrawal, increased after 21 d withdrawal, and returned to the basal level upon cocaine re-exposure after 21 d withdrawal. These results, together with previous results showing cocaine-induced adaptations at excitatory synapses and intrinsic membrane excitability of NAc MSNs, may provide a relatively thorough picture of the functional state of NAc MSNs following cocaine exposure.

Homeostatic recovery of downstate–upstate cycling in nucleus accumbens neurons

Homeostatic plasticity is a powerful cellular mechanism through which neurons adjust intracellular and intercellular resources to stabilize their functional output through the ever-changing environment. Here, we report a novel form of homeostatic plasticity that nucleus accumbens (NAc) neurons use to regain their functionally active state once it is lost. In vivo, NAc neurons periodically alternate between a functionally active upstate and a functionally quiescent downstate. The upstate of NAc neurons is immediately lost following severe environmental changes, such as deep anesthesia and truncation of excitatory synaptic inputs. Using short-term slice cultures, our current study demonstrates that NAc neurons initially lose but gradually recover their upstate-downstate cycling after shifting to the in vitro condition. Furthermore, we show that this homeostatic recovery of the upstate is mediated by increased synchronization of presynaptic activity. Given that being in the upstate is required for in vivo NAc neurons to fire action potentials, the homeostatic recovery of upstate may underlie an important cellular mechanism for NAc neurons to maintain their functional output against severe environmental fluctuations.