Giannina Descalzi

Alleviating Neuropathic Pain Hypersensitivity by Inhibiting PKMζ in the Anterior Cingulate Cortex

Pain in the Brain One of the major challenges in pain research is finding ways to reverse chronic pain. Synaptic long-term potentiation (LTP) at spinal or cortical levels is a cellular model of chronic pain. X.-Y. Li. et al. (p. 1400) studied the role of the enzyme protein kinase M zeta (PKMζ) in neurons of the anterior cingulate cortex (ACC) in the maintenance of LTP and for enhanced pain sensitivity after peripheral nerve injury in mice. Nerve injury appeared to lead to the up-regulation and phosphorylation of PKMζ. This triggered LTP at some synapses in the ACC by increasing the number of AMPA receptors. LTP was restricted to ACC neurons that were activated by nerve injury. Blocking PKMζ in the ACC days after nerve injury normalized pain behavior. Thus, PKMζ may represent a promising target for the treatment of chronic pain. Nerve injury increases the activity of an enzyme in the brain and contributes to chronic pain–related cortical sensitization. Synaptic plasticity is a key mechanism for chronic pain. It occurs at different levels of the central nervous system, including spinal cord and cortex. Studies have mainly focused on signaling proteins that trigger these plastic changes, whereas few have addressed the maintenance of plastic changes related to chronic pain. We found that protein kinase M zeta (PKMζ) maintains pain-induced persistent changes in the mouse anterior cingulate cortex (ACC). Peripheral nerve injury caused activation of PKMζ in the ACC, and inhibiting PKMζ by a selective inhibitor, ζ-pseudosubstrate inhibitory peptide (ZIP), erased synaptic potentiation. Microinjection of ZIP into the ACC blocked behavioral sensitization. These results suggest that PKMζ in the ACC acts to maintain neuropathic pain. PKMζ could thus be a new therapeutic target for treating chronic pain.

Sleep deprivation impairs cAMP signalling in the hippocampus

Millions of people regularly obtain insufficient sleep. Given the effect of sleep deprivation on our lives, understanding the cellular and molecular pathways affected by sleep deprivation is clearly of social and clinical importance. One of the major effects of sleep deprivation on the brain is to produce memory deficits in learning models that are dependent on the hippocampus. Here we have identified a molecular mechanism by which brief sleep deprivation alters hippocampal function. Sleep deprivation selectively impaired 3′, 5′-cyclic AMP (cAMP)- and protein kinase A (PKA)-dependent forms of synaptic plasticity in the mouse hippocampus, reduced cAMP signalling, and increased activity and protein levels of phosphodiesterase 4 (PDE4), an enzyme that degrades cAMP. Treatment of mice with phosphodiesterase inhibitors rescued the sleep-deprivation-induced deficits in cAMP signalling, synaptic plasticity and hippocampus-dependent memory. These findings demonstrate that brief sleep deprivation disrupts hippocampal function by interfering with cAMP signalling through increased PDE4 activity. Thus, drugs that enhance cAMP signalling may provide a new therapeutic approach to counteract the cognitive effects of sleep deprivation.

Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain

Chronic pain can lead to anxiety and anxiety can enhance the sensation of pain. Unfortunately, little is known about the synaptic mechanisms that mediate these re-enforcing interactions. Here we characterized two forms of long-term potentiation (LTP) in the anterior cingulate cortex (ACC); a presynaptic form (pre-LTP) that requires kainate receptors and a postsynaptic form (post-LTP) that requires N-methyl-D-aspartate receptors. Pre-LTP also involves adenylyl cyclase and protein kinase A and is expressed via a mechanism involving hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Interestingly, chronic pain and anxiety both result in selective occlusion of pre-LTP. Significantly, microinjection of the HCN blocker ZD7288 into the ACC in vivo produces both anxiolytic and analgesic effects. Our results provide a mechanism by which two forms of LTP in the ACC may converge to mediate the interaction between anxiety and chronic 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.

An Increase in Synaptic NMDA Receptors in the Insular Cortex Contributes to Neuropathic Pain

Inhibiting NMDA receptor function in the insular cortex may prevent the development of neuropathic pain. Stopping the Pain Damage to the central or peripheral nervous system can trigger the development of neuropathic pain, which can manifest as painful sensations in response to stimuli that are not normally painful. Qiu et al. found that mice that had developed neuropathic pain after peripheral nerve injury showed changes in synaptic plasticity and increased abundance of synaptic NMDA receptors in the insular cortex, a region of the brain that is activated by acute and chronic pain. Using pharmacological inhibitors and transgenic mice, they mimicked these changes in vitro with insular cortical slices and thus identified the signaling pathway responsible. Mice injected with NMDA receptor inhibitors showed reduced behavioral signs of neuropathic pain after peripheral nerve injury. Thus, blocking NMDA receptor function in the insular cortex may prevent the development of neuropathic pain. Neurons in the insular cortex are activated by acute and chronic pain, and inhibition of neuronal activity in the insular cortex has analgesic effects. We found that in a mouse model in which peripheral nerve injury leads to the development of neuropathic pain, the insular cortex showed changes in synaptic plasticity, which were associated with a long-term increase in the amount of synaptic N-methyl-d-aspartate receptors (NMDARs), but not that of extrasynaptic NMDARs. Activation of cyclic adenosine monophosphate (cAMP)–dependent signaling enhanced the amount of synaptic NMDARs in acutely isolated insular cortical slices and increased the surface localization of NMDARs in cultured cortical neurons. We found that the increase in the amount of NMDARs required phosphorylation of the NMDAR subunit GluN2B at Tyr1472 by a pathway involving adenylyl cyclase subtype 1 (AC1), protein kinase A (PKA), and Src family kinases. Finally, injecting NMDAR or GluN2B-specific antagonists into the insular cortex reduced behavioral responses to normally nonnoxious stimuli in the mouse model of neuropathic pain. Our results suggest that activity-dependent plasticity takes place in the insular cortex after nerve injury and that inhibiting the increase in NMDAR function may help to prevent or treat neuropathic pain.

Glutamate acts as a neurotransmitter for gastrin releasing peptide-sensitive and insensitive itch-related synaptic transmission in mammalian spinal cord

Itch sensation is one of the major sensory experiences of human and animals. Recent studies have proposed that gastrin releasing peptide (GRP) is a key neurotransmitter for itch in spinal cord. However, no direct evidence is available to indicate that GRP actually mediate responses between primary afferent fibers and dorsal horn neurons. Here we performed integrative neurobiological experiments to test this question. We found that a small population of rat dorsal horn neurons responded to GRP application with increases in calcium signaling. Whole-cell patch-clamp recordings revealed that a part of superficial dorsal horn neurons responded to GRP application with the increase of action potential firing in adult rats and mice, and these dorsal horn neurons received exclusively primary afferent C-fiber inputs. On the other hands, few Aδ inputs receiving cells were found to be GRP positive. Finally, we found that evoked sensory responses between primary afferent C fibers and GRP positive superficial dorsal horn neurons are mediated by glutamate but not GRP. CNQX, a blocker of AMPA and kainate (KA) receptors, completely inhibited evoked EPSCs, including in those Fos-GFP positive dorsal horn cells activated by itching. Our findings provide the direct evidence that glutamate is the principal excitatory transmitter between C fibers and GRP positive dorsal horn neurons. Our results will help to understand the neuronal mechanism of itch and aid future treatment for patients with pruritic disease.

Neurabin in the anterior cingulate cortex regulates anxiety-like behavior in adult mice

Affective disorders, which include anxiety and depression, are highly prevalent and have overwhelming emotional and physical symptoms. Despite human brain imaging studies, which have implicated the prefrontal cortex including the anterior cingulate cortex (ACC), little is known about the ACC in anxiety disorders. Here we show that the ACC does modulate anxiety-like behavior in adult mice, and have identified a protein that is critical for this modulation. Absence of neurabin, a cytoskeletal protein, resulted in reduced anxiety-like behavior and increased depression-like behavior. Selective inhibition of neurabin in the ACC reproduced the anxiety but not the depression phenotype. Furthermore, loss of neurabin increased the presynaptic release of glutamate and cingulate neuronal excitability. These findings reveal novel roles of the ACC in anxiety disorders, and provide a new therapeutic target for the treatment of anxiety disorders.

Postsynaptic potentiation of corticospinal projecting neurons in the anterior cingulate cortex after nerve injury

Long-term potentiation (LTP) is the key cellular mechanism for physiological learning and pathological chronic pain. In the anterior cingulate cortex (ACC), postsynaptic recruitment or modification of AMPA receptor (AMPAR) GluA1 contribute to the expression of LTP. Here we report that pyramidal cells in the deep layers of the ACC send direct descending projecting terminals to the dorsal horn of the spinal cord (lamina I-III). After peripheral nerve injury, these projection cells are activated, and postsynaptic excitatory responses of these descending projecting neurons were significantly enhanced. Newly recruited AMPARs contribute to the potentiated synaptic transmission of cingulate neurons. PKA-dependent phosphorylation of GluA1 is important, since enhanced synaptic transmission was abolished in GluA1 phosphorylation site serine-845 mutant mice. Our findings provide strong evidence that peripheral nerve injury induce long-term enhancement of cortical-spinal projecting cells in the ACC. Direct top-down projection system provides rapid and profound modulation of spinal sensory transmission, including painful information. Inhibiting cortical top-down descending facilitation may serve as a novel target for treating neuropathic pain.

Translational investigation and treatment of neuropathic pain

Neuropathic pain develops from a lesion or disease affecting the somatosensory system. Translational investigations of neuropathic pain by using different animal models reveal that peripheral sensitization, spinal and cortical plasticity may play critical roles in neuropathic pain. Furthermore, descending facilitatory or excitatory modulation may also act to enhance chronic pain. Current clinical therapy for neuropathic pain includes the use of pharmacological and nonpharmacological (psychological, physical, and surgical treatment) methods. However, there is substantial need to better medicine for treating neuropathic pain. Future translational researchers and clinicians will greatly facilitate the development of novel drugs for treating chronic pain including neuropathic pain.

Fragile X mental retardation protein in learning-related synaptic plasticity

Fragile X syndrome (FXS) is caused by a lack of the fragile X mental retardation protein (FMRP) due to silencing of the Fmr1 gene. As an RNA binding protein, FMRP is thought to contribute to synaptic plasticity by regulating plasticity-related protein synthesis and other signaling pathways. Previous studies have mostly focused on the roles of FMRP within the hippocampus - a key structure for spatial memory. However, recent studies indicate that FMRP may have a more general contribution to brain functions, including synaptic plasticity and modulation within the prefrontal cortex. In this brief review, we will focus on recent studies reported in the prefrontal cortex, including the anterior cingulate cortex (ACC). We hypothesize that alterations in ACC-related plasticity and synaptic modulation may contribute to various forms of cognitive deficits associated with FXS.