Xue-Han Zhang

Roles of NMDA NR2B Subtype Receptor in Prefrontal Long-Term Potentiation and Contextual Fear Memory

Cortical plasticity is thought to be important for the establishment, consolidation, and retrieval of permanent memory. Hippocampal long-term potentiation (LTP), a cellular mechanism of learning and memory, requires the activation of glutamate N-methyl-D-aspartate (NMDA) receptors. In particular, it has been suggested that NR2A-containing NMDA receptors are involved in LTP induction, whereas NR2B-containing receptors are involved in LTD induction in the hippocampus. However, LTP in the prefrontal cortex is less well characterized than in the hippocampus. Here we report that the activation of the NR2B and NR2A subunits of the NMDA receptor is critical for the induction of cingulate LTP, regardless of the induction protocol. Furthermore, pharmacological or genetic blockade of the NR2B subunit in the cingulate cortex impaired the formation of early contextual fear memory. Our results demonstrate that the NR2B subunit of the NMDA receptor in the prefrontal cortex is critically involved in both LTP and contextual memory.

Presynaptic and postsynaptic amplifications of neuropathic pain in the anterior cingulate cortex.

Neuropathic pain is caused by a primary lesion or dysfunction in the nervous system. Investigations have mainly focused on the spinal mechanisms of neuropathic pain, and less is known about cortical changes in neuropathic pain. Here, we report that peripheral nerve injury triggered long-term changes in excitatory synaptic transmission in layer II/III neurons within the anterior cingulate cortex (ACC). Both the presynaptic release probability of glutamate and postsynaptic glutamate AMPA receptor-mediated responses were enhanced after injury using the mouse peripheral nerve injury model. Western blot showed upregulated phosphorylation of GluR1 in the ACC after nerve injury. Finally, we found that both presynaptic and postsynaptic changes after nerve injury were absent in genetic mice lacking calcium-stimulated adenylyl cyclase 1 (AC1). Our studies therefore provide direct integrative evidence for both long-term presynaptic and postsynaptic changes in cortical synapses after nerve injury, and that AC1 is critical for such long-term changes. AC1 thus may serve as a potential therapeutic target for treating neuropathic pain.

Identification of an Adenylyl Cyclase Inhibitor for Treating Neuropathic and Inflammatory Pain

In animal models, an adenylyl cyclase 1 inhibitor acts as an effective treatment for neuropathic pain, in part by acting on the anterior cingulate cortex. No Gain from Pain Pain from a hot stove or an injury can be a good thing. It can help to prevent more serious damage, but chronic, burning, or aching pain—also called neuropathic pain—seems to have no purpose. Analgesics that block only neuropathic pain are desirable but scarce. Wang et al. have now identified a promising new candidate by screening for drugs that selectively block a type of calcium-activated adenylyl cyclase that participates in neuropathic pain. They identify one, NB001, which can block this type of pain in rodents without apparent side effects. Adenylyl cyclase 1 has many characteristics of a good drug target for neuropathic pain: It is an activity-dependent enzyme, expressed selectively in neurons, that is critical for the pain-related neural plasticity thought to underlie this kind of pain. The authors screened chemical compounds for inhibition of cyclic AMP production and of the transcription factor CREB in human cells transfected with adenylyl cyclase 1. One of these, NB001, was most effective and also inhibited cyclic AMP production in mouse brain slices and human neurons. NB001 prevented allodynia (a condition in which an innocuous stimulus causes pain) in mice in which certain nerves were ligated or in mice with chronic inflammatory pain, produced by an injection of an irritant into a paw. And when the drug was injected directly into the anterior cingulate cortex (a brain region involved in neuropathic pain generation), it also prevented allodynia, although to a lesser extent, suggesting that NB001 acts on multiple sites in the body. Just as important as these effects of NB001 on chronic pain are the effects that it does not have. NB001 does not interfere with normal nociception, the sensation that allows the animal to escape dangerous heat. It does not affect neurotransmission of the critical hormone glutamate or the size of glutamate-induced currents. Tests of anxiety, motor function, and fear all showed that NB001 had no effects on these endpoints, a good sign for the potential safety profile of this drug. A clue to how NB001 works can be gleaned from the result that it extinguishes the ability of synapses in the dorsal horn of the spinal cord and the anterior cingulate cortex to “learn,” a process triggered in neuropathic pain. This effect may underlie its analgesic ability, a conclusion consistent with the fact that it does not alter such plasticity in the hippocampus, a non–pain-related brain region. If the selective action of NB001 on neuropathic pain and its lack of serious side effects also holds true in humans, it may prove useful to eliminating seemingly purposeless pain from our lives. Neuropathic pain, often caused by nerve injury, is commonly observed among patients with different diseases. Because its basic mechanisms are poorly understood, effective medications are limited. Previous investigations of basic pain mechanisms and drug discovery efforts have focused mainly on early sensory neurons such as dorsal root ganglion and spinal dorsal horn neurons, and few synaptic-level studies or new drugs are designed to target the injury-related cortical plasticity that accompanies neuropathic pain. Our previous work has demonstrated that calcium-stimulated adenylyl cyclase 1 (AC1) is critical for nerve injury–induced synaptic changes in the anterior cingulate cortex. Through rational drug design and chemical screening, we have identified a lead candidate AC1 inhibitor, NB001, which is relatively selective for AC1 over other adenylate cyclase isoforms. Using a variety of behavioral tests and toxicity studies, we have found that NB001, when administered intraperitoneally or orally, has an analgesic effect in animal models of neuropathic pain, without any apparent side effects. Our study thus shows that AC1 could be a productive therapeutic target for neuropathic pain and describes a new agent for the possible treatment of neuropathic pain.

Increased Anxiety-Like Behavior and Enhanced Synaptic Efficacy in the Amygdala of GluR5 Knockout Mice

GABAergic transmission in the amygdala modulates the expression of anxiety. Understanding the interplay between GABAergic transmission and excitatory circuits in the amygdala is, therefore, critical for understanding the neurobiological basis of anxiety. Here, we used a multi-disciplinary approach to demonstrate that GluR5-containing kainate receptors regulate local inhibitory circuits, modulate the excitatory transmission from the basolateral amygdala to the central amygdala, and control behavioral anxiety. Genetic deletion of GluR5 or local injection of a GluR5 antagonist into the basolateral amygdala increases anxiety-like behavior. Activation of GluR5 selectively depolarized inhibitory neurons, thereby increasing GABA release and contributing to tonic GABA current in the basolateral amygdala. The enhanced GABAergic transmission leads to reduced excitatory inputs in the central amygdala. Our results suggest that GluR5 is a key regulator of inhibitory circuits in the amygdala and highlight the potential use of GluR5-specific drugs in the treatment of pathological anxiety.

Differential regulation of β-arrestin 1 and β-arrestin 2 gene expression in rat brain by morphine

Abstract β-Arrestins are a family of regulatory and scaffold proteins functioning in signal transduction of G protein-coupled receptors including opioid receptors. Upon agonist stimulation, β-arrestins bind to opioid receptors phosphorylated by G protein-coupled receptor kinases and promote receptor internalization and desensitization. Studies indicated that β-arrestins are required in the development of morphine tolerance in mice. In the current study, we investigated the potential regulatory effects of morphine administration on β-arrestin 1 and β-arrestin 2 mRNA levels in different brain regions in rat using in situ hybridization method. Our results showed that the acute morphine administration (10 mg/kg) resulted in approximately 30% reduction in both β-arrestin 1 and β-arrestin 2 mRNA levels in hippocampus while the chronic morphine treatment (10 mg/kg, b.i.d., for 9 days) caused no significant change in level of either β-arrestin mRNA. In locus coeruleus, both acute and chronic morphine treatments resulted in significant decreases (over 50%) in β-arrestin 1 mRNA level but failed to induce any change in the level of β-arrestin 2 gene expression. The acute morphine administration had no significant effect on β-arrestin 1 or β-arrestin 2 mRNA level in periaqueductal gray and cerebral cortex. However, after chronic morphine treatment, β-arrestin 2 mRNA level decreased by 40% in periaqueductal gray and increased by 25% in cerebral cortex, in strong contrast to the unchanged β-arrestin 1 mRNA level in these two brain regions. Furthermore, spontaneous or naloxone-precipitated withdrawal of morphine that did not affect the level of β-arrestin 1 mRNA resulted in an aberrant increase (100% over control) in β-arrestin 2 mRNA level in hippocampus. Our results thus demonstrated for the first time that opiate administration regulates level of β-arrestin mRNAs in brain and the expression of β-arrestin 1 and β-arrestin 2 subtypes is differentially regulated in locus coeruleus, periaqueductal gray, and cerebral cortex by morphine. These data suggest that β-arrestin 1 and β-arrestin 2 may play different roles in the development of opioid tolerance and dependence.

Acute and chronic morphine treatments and morphine withdrawal differentially regulate GRK2 and GRK5 gene expression in rat brain.

Opioid agonist stimulates activation of G protein-coupled receptor kinase (GRK) and causes desensitization of opioid signaling, which plays an important role in opioid tolerance. The current study investigated the potential regulatory effects of acute and chronic morphine administration and withdrawal on GRK2 and GRK5 gene expression in rat brain. Our results showed that the initial morphine treatment (10 mg/kg) significantly increased GRK mRNA levels in cerebral cortex, hippocampus, and lateral thalamic nuclei. A significant decrease in GRK5 mRNA levels was observed in periaqueductal gray. In strong contrast, repeated administration of morphine for 9 days failed to cause any significant increase in GRK5 mRNA in any of these brain regions. Chronic morphine treatment resulted in 30-70% down-regulation of GRK2 expression in cerebral cortex, hippocampus, thalamus, and locus coeruleus, opposite to what observed with the single morphine administration. Moreover, spontaneous and naloxone-precipitated morphine withdrawal resulted in aberrant increases in GRK2 and GRK5 mRNA levels in these brain regions. Taken together, our study suggests that opioid not only induces rapid negative feedback regulation on opioid signals through activation of GRK but also exerts its impact, via controlling levels of GRK gene expression, on the regulatory machinery itself over a longer period of time in brain.

Induction- and conditioning-protocol dependent involvement of NR2B-containing NMDA receptors in synaptic potentiation and contextual fear memory in the hippocampal CA1 region of rats

Long-term potentiation (LTP) in the hippocampal CA1 region requires the activation of N-methyl-D-aspartate receptors (NMDARs). Studies using genetic and pharmacological approaches have reported inconsistent results of the requirement of NR2B-containing NMDARs in LTP in the CA1 region. Pharmacological studies showed that NR2B-containing NMDARs are not required for LTP, while genetic studies reported that over-expression of NR2B-NMDARs enhances LTP and hippocampus-dependent memory. Here, we provide evidence showing that the functional role of NR2B-NMDARs in hippocampal LTP and memory depends on LTP-inducing and behavior-conditioning protocols. Inhibition of NR2B-NMDARs with the NR2B selective antagonist ifenprodil or Ro25-6981 suppressed LTP induced by spike-timing protocol, with no impact on LTP induced by pairing protocol or two-train high-frequency stimulation (HFS) protocol. Inhibition of NR2B-NMDARs did not affect the late phase LTP induced by four-train HFS. Ca2+ imaging showed that there was difference in kinetics of intracellular Ca2+ signals induced by spiking-timing and pairing protocols. Pre-training intra-CA1 infusion of ifenprodil or Ro25-6981 impaired the contextual fear memory induced by five CS-US pairings, with no effect on the memory induced by one CS-US pairing.

Genetic enhancement of trace fear memory and cingulate potentiation in mice overexpressing Ca2+/calmodulin-dependent protein kinase IV

Long‐term potentiation (LTP) is a key cellular model for studying mechanisms for learning and memory. Previous studies reported that the Ca2+/calmodulin‐dependent protein kinase IV (CaMKIV) is critical for gene regulation, and behavioral learning and memory. Less is known about the roles of CaMKIV in cortical plasticity and trace fear memory. Here we have found that LTP was significantly enhanced in the anterior cingulate cortex (ACC) of the mice overexpressing CaMKIV. By contrast, neither α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor‐mediated basal excitatory synaptic transmission nor N‐methyl‐d‐aspartate (NMDA) receptor‐mediated excitatory postsynaptic currents were affected. Furthermore, paired‐pulse ratio in the transgenic mice is normal. In behavioral tests, we found that the CaMKIV transgenic mice exhibited significant enhancement in trace fear memory, while the acute sensory thresholds were not affected. Our results provide strong evidence that forebrain CaMKIV contributes to trace fear memory by enhancing synaptic potentiation in the ACC.

Chronic morphine treatment and withdrawal induce up-regulation of c-Jun N-terminal kinase 3 gene expression in rat brain.

Chronic opiate applications produce long-term impacts on many functions of the brain and induce tolerance, dependence, and addiction. It has been demonstrated that opioid drugs are capable to induce apoptosis of neuronal cells, but the mechanism is not clear. c-Jun N-terminal kinase 3 (JNK3), specifically expressed in brain, has been proved to mediate neuronal apoptosis and is involved in opiate-induced cell apoptosis in vitro. The present study investigated the effect of opioid administration on expression of JNK3, an important mediator involved in apoptosis of neurons, in rat brain. Our results showed that single or chronic injection of morphine resulted in a 45-50% increase in the level of JNK3 mRNA in frontal cortex, while no significant change was detected in other brain regions such as thalamus, hippocampus and locus coeruleus. Similar to what was observed after the acute or chronic morphine administration, no significant change in JNK3 expression was detected in locus coeruleus following cessation of the chronic morphine administration. However, interestingly, sustained elevation of JNK3 expression peaked on day 14 after cessation of morphine treatment was observed in the brain regions such as hippocampus and thalamus, where acute or chronic morphine treatment did not cause any significant change in JNK3 gene expression. The increased JNK3 mRNA in these brain areas returned to the control levels in 28 days following cessation of chronic morphine treatment. Taken together, these results demonstrated for the first time that the expression of JNK3 gene is regulated by opioids and that chronic opioid administration and withdrawal could induce sustained elevation of JNK3 mRNA in many important brain areas. The changes in JNK3 gene expression in brain induced by chronic opioid treatment may play a role in opioid-induced apoptosis and neurotoxicity.

Conditioning-strength dependent involvement of NMDA NR2B subtype receptor in the basolateral nucleus of amygdala in acquisition of auditory fear memory.

It is known that N-methyl-D-aspartate (NMDA) receptor in the basolateral nucleus of amygdala (BLA) is essential for fear memory formation. NMDA NR2B and NR2A subtype receptors exhibit difference in electrophysiological and signaling properties. However, it is unclear whether these two subtype receptors have different roles in fear memory formation. Here, we provide evidence, using pharmacological blockade and genetic interference, that NR2B is involved in acquisition of auditory fear memory in a conditioning-strength dependent way. Pre-conditioning intra-BLA infusion of the NR2B selective antagonist ifenprodil or Ro25-6981 impaired 48-h auditory fear memory (AFM) induced by five but not one CS-US pairing protocol, while similar treatment with the NR2A antagonist NVP-AAM077 disrupted memory for both protocols. Consistently, genetic over-expression of NR2B C-terminal in the BLA, which interferes with the C-terminal mediated intracellular signaling, produced a severe deficit in 48-h AFM for five but not one CS-US pairing protocol, whereas over-expression of NR2A C-terminal impaired memory for both protocols. Furthermore, pre-conditioning infusion of ifenprodil down-regulated the elevated phosphorylation level of extracellular signal-regulated kinase (ERK) induced by five CS-US pairing protocol. Thus, the involvement of BLA NR2B in AFM acquisition depends on conditioning strength.