Kunjumon I. Vadakkan

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.

Calcium Calmodulin-Stimulated Adenylyl Cyclases Contribute to Activation of Extracellular Signal-Regulated Kinase in Spinal Dorsal Horn Neurons in Adult Rats and Mice

The extracellular signal-regulated kinase (Erk) cascades are suggested to contribute to excitatory synaptic plasticity in the CNS, including the spinal cord dorsal horn. However, many of their upstream signaling pathways remain to be investigated. Here, we demonstrate that glutamate and substance P (SP), two principal mediators of sensory information between primary afferent fibers and the spinal cord, activate Erk in dorsal horn neurons of both adult rat and mouse spinal cord. In genetic knock-out mice of calcium calmodulin-stimulated adenylyl cyclase subtypes 1 (AC1) and 8 (AC8), activation of Erk in dorsal horn neurons were significantly reduced or blocked, either after peripheral tissue inflammation or by glutamate or SP in spinal cord slices. Our studies suggest that AC1 and AC8 act upstream from Erk activation in spinal dorsal horn neurons and the calcium-AC1/AC8-dependent Erk signaling pathways may contribute to spinal sensitization, an underlying mechanism for the development of persistent pain after injury.

ATP-induced chemotaxis of microglial processes requires P2Y receptor-activated initiation of outward potassium currents.

Microglial cells are the resident macrophages that are involved in brain injuries and infections. Recent studies using transcranial two‐photon microscopy have shown that ATP and P2Y receptors mediated rapid chemotactic responses of miroglia to local injury. However, the molecular mechanism for microglial chemotaxis toward ATP is still unknown. To address this question, we employed a combination of simultaneous perforated whole‐cell recordings and time‐lapse confocal imaging in GFP‐labeled microglia in acute brain slices from adult mice. We found that ATP‐induced rapid chemotaxis is correlated with P2Y receptor associated‐outward potassium current in microglia. Activation of both P2Y receptor and its associated potassium channels are required for ATP‐induced chemotaxis and baseline motility of microglial cells. The chemotaxis required the activation of phosphoinositide 3‐kinase but not mitogen‐activated protein kinase pathway. Our results provide strong evidence that P2Y receptor‐associated outward potassium channels and the phosphoinositide 3‐kinase pathway are important for ATP‐induced microglial motility in acute brain slices.

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.

Genetic Evidence for Adenylyl Cyclase 1 as a Target for Preventing Neuronal Excitotoxicity Mediated by N-Methyl-D-aspartate Receptors

The excessive activation of N-methyl-d-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.

Genetic reduction of chronic muscle pain in mice lacking calcium/calmodulin-stimulated adenylyl cyclases

BackgroundThe Ca2+/calmodulin-stimulated adenylyl cyclase (AC) isoforms AC1 and AC8, couple NMDA receptor activation to cAMP signaling pathways in neurons and are important for development, learning and memory, drug addiction and persistent pain. AC1 and AC8 in the anterior cingulate cortex (ACC) and the spinal cord were previously shown to be important in subcutaneous inflammatory pain. Muscle pain is different from cutaneous pain in its characteristics as well as conducting fibers. Therefore, we conducted the present work to test the role of AC1 and AC8 in both acute persistent and chronic muscle pain.ResultsUsing an acute persistent inflammatory muscle pain model, we found that the behavioral nociceptive responses of both the late phase of acute muscle pain and the chronic muscle inflammatory pain were significantly reduced in AC1 knockout (KO) and AC1&8 double knockout (DKO) mice. Activation of other adenylyl cyclases in these KO mice by microinjection of forskolin into the ACC or spinal cord, but not into the peripheral tissue, rescued the behavioral nociceptive responses. Additionally, intra-peritoneal injection of an AC1 inhibitor significantly reduced behavioral responses in both acute persistent and chronic muscle pain.ConclusionThe results of the present study demonstrate that neuronal Ca2+/calmodulin-stimulated adenylyl cyclases in the ACC and spinal cord are important for both late acute persistent and chronic inflammatory muscle pain.

Presynaptic regulation of the inhibitory transmission by GluR5-containing kainate receptors in spinal substantia gelatinosa.

GluR5-containing kainate receptors (KARs) are known to be involved in nociceptive transmission. Our previous work has shown that the activation of presynaptic KARs regulates GABAergic and glycinergic synaptic transmission in cultured dorsal horn neurons. However, the role of GluR5-containing KARs in the modulation of inhibitory transmission in the spinal substantia gelatinosa (SG) in slices remains unknown. In the present study, pharmacological, electrophysiological and genetic methods were used to show that presynaptic GluR5 KARs are involved in the modulation of inhibitory transmission in the SG of spinal slices in vitro. The GluR5 selective agonist, ATPA, facilitated the frequency but not amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) in SG neurons. ATPA increased sIPSC frequency in all neurons with different firing patterns as delayed, tonic, initial and single spike patterns. The frequency of either GABAergic or glycinergic sIPSCs was significantly increased by ATPA. ATPA could also induce inward currents in all SG neurons recorded. The frequency, but not amplitude, of action potential-independent miniature IPSCs (mIPSCs) was also facilitated by ATPA in a concentration-dependent manner. However, the effect of ATPA on the frequency of either sIPSCs or mIPSCs was abolished in GluR5-/- mice. Deletion of the GluR5 subunit gene had no effect on the frequency or amplitude of mIPSCs in SG neurons. However, GluR5 antagonist LY293558 reversibly inhibited sIPSC and mIPSC frequencies in spinal SG neurons. Taken together, these results suggest that GluR5 KARs, which may be located at presynaptic terminals, contribute to the modulation of inhibitory transmission in the SG. GluR5-containing KARs are thus important for spinal sensory transmission/modulation in the spinal cord.

Cell-type specific proximity of centromeric domains of one homologue each of chromosomes 2 and 11 in nuclei of cerebellar Purkinje neurons

In Purkinje neurons of the mouse cerebellum, the centromeres of several chromosomes are placed in close proximity to form a distinct pattern of clusters and exhibit reproducible spatial redistributions during development. In granule neurons, an adjacent cell type in the cerebellum, the pattern, size, and number of centromeric aggregations are different from those of Purkinje neurons. The present work was undertaken to test the hypothesis that the same chromosomes form part of one aggregate in a cell-type-specific manner. Fluorescence in situ hybridization (FISH) with chromosome-specific paracentromeric probes was used to identify centromeric regions of individual chromosomes in cerebellar Purkinje and granule neurons of the adult mouse. When pairs of centromeric probes were used in two-color FISH, one homologue each of chromosomes 2 and 11 were routinely found close to each other in Purkinje neurons but not in granule neurons. This finding of specific proximity was limited to the pair 2 and 11, out of the ten chromosome pairs that were randomly selected and studied. Our results indicate that, in adult Purkinje neurons, a cell-type-specific spatial proximity is present between centromeric domains of one homologue each of chromosomes 2 and 11.

Trend towards varying combinatorial centromere association in morphologically identical clusters in Purkinje neurons

Neurons with similar morphology and neurotransmitter content located at a specific brain region may be part of the same or functionally separate networks. To address the question whether morphologically similar neurons have similar structural architecture at the chromosomal level, we studied Purkinje neurons in the cerebellum. Previous studies have shown that in Purkinje neurons centromeres of several chromosomes form clusters and that the number and size of these clusters remain stable in the adult brain. We examined whether the same set of centromeres form clusters in all the Purkinje neurons. Fluorescent in situ hybridization (FISH) with chromosome-specific para-centromeric probes provided an indirect evidence for a trend towards varying contributions from different chromosomes forming the centromeric clusters in adjacent Purkinje neurons. The results of the study indicate that the individual Purkinje neurons are likely unique in inter-chromosomal spatial associations.