Working memory impairment in calcineurin knock-out mice is associated with alterations in synaptic vesicle cycling and disruption of high-frequency synaptic and network activity in prefrontal cortex.

Working memory is an essential component of higher cognitive function, and its impairment is a core symptom of multiple CNS disorders, including schizophrenia. Neuronal mechanisms supporting working memory under normal conditions have been described and include persistent, high-frequency activity of prefrontal cortical neurons. However, little is known about the molecular and cellular basis of working memory dysfunction in the context of neuropsychiatric disorders. To elucidate synaptic and neuronal mechanisms of working memory dysfunction, we have performed a comprehensive analysis of a mouse model of schizophrenia, the forebrain-specific calcineurin knock-out mouse. Biochemical analyses of cortical tissue from these mice revealed a pronounced hyperphosphorylation of synaptic vesicle cycling proteins known to be necessary for high-frequency synaptic transmission. Examination of the synaptic vesicle cycle in calcineurin-deficient neurons demonstrated an impairment of vesicle release enhancement during periods of intense stimulation. Moreover, brain slice and in vivo electrophysiological analyses showed that loss of calcineurin leads to a gene dose-dependent disruption of high-frequency synaptic transmission and network activity in the PFC, correlating with selective working memory impairment. Finally, we showed that levels of dynamin I, a key presynaptic protein and calcineurin substrate, are significantly reduced in prefrontal cortical samples from schizophrenia patients, extending the disease relevance of our findings. Our data provide support for a model in which impaired synaptic vesicle cycling represents a critical node for disease pathologies underlying the cognitive deficits in schizophrenia.

TRPA1 is a major oxidant sensor in murine airway sensory neurons.

Sensory neurons in the airways are finely tuned to respond to reactive chemicals threatening airway function and integrity. Nasal trigeminal nerve endings are particularly sensitive to oxidants formed in polluted air and during oxidative stress as well as to chlorine, which is frequently released in industrial and domestic accidents. Oxidant activation of airway neurons induces respiratory depression, nasal obstruction, sneezing, cough, and pain. While normally protective, chemosensory airway reflexes can provoke severe complications in patients affected by inflammatory airway conditions like rhinitis and asthma. Here, we showed that both hypochlorite, the oxidizing mediator of chlorine, and hydrogen peroxide, a reactive oxygen species, activated Ca(2+) influx and membrane currents in an oxidant-sensitive subpopulation of chemosensory neurons. These responses were absent in neurons from mice lacking TRPA1, an ion channel of the transient receptor potential (TRP) gene family. TRPA1 channels were strongly activated by hypochlorite and hydrogen peroxide in primary sensory neurons and heterologous cells. In tests of respiratory function, Trpa1(-/-) mice displayed profound deficiencies in hypochlorite- and hydrogen peroxide-induced respiratory depression as well as decreased oxidant-induced pain behavior. Our results indicate that TRPA1 is an oxidant sensor in sensory neurons, initiating neuronal excitation and subsequent physiological responses in vitro and in vivo.

Transient receptor potential ankyrin 1 antagonists block the noxious effects of toxic industrial isocyanates and tear gases.

The release of methyl isocyanate in Bhopal, India, caused the worst industrial accident in history. Exposures to industrial isocyanates induce lacrimation, pain, airway irritation, and edema. Similar responses are elicited by chemicals used as tear gases. Despite frequent exposures, the biological targets of isocyanates and tear gases in vivo have not been identified, precluding the development of effective countermeasures. We use Ca(2+) imaging and electrophysiology to show that the noxious effects of isocyanates and those of all major tear gas agents are caused by activation of Ca(2+) influx and membrane currents in mustard oil-sensitive sensory neurons. These responses are mediated by transient receptor potential ankyrin 1 (TRPA1), an ion channel serving as a detector for reactive chemicals. In mice, genetic ablation or pharmacological inhibition of TRPA1 dramatically reduces isocyanate- and tear gas-induced nocifensive behavior after both ocular and cutaneous exposures. We conclude that isocyanates and tear gas agents target the same neuronal receptor, TRPA1. Treatment with TRPA1 antagonists may prevent and alleviate chemical irritation of the eyes, skin, and airways and reduce the adverse health effects of exposures to a wide range of toxic noxious chemicals.

Chronic exposure to paclitaxel diminishes phosphoinositide signaling by calpain-mediated neuronal calcium sensor-1 degradation.

Paclitaxel (Taxol) is a well established chemotherapeutic agent for the treatment of solid tumors, but it is limited in its usefulness by the frequent induction of peripheral neuropathy. We found that prolonged exposure of a neuroblastoma cell line and primary rat dorsal root ganglia with therapeutic concentrations of Taxol leads to a reduction in inositol trisphosphate (InsP(3))-mediated Ca(2+) signaling. We also observed a Taxol-specific reduction in neuronal calcium sensor 1 (NCS-1) protein levels, a known modulator of InsP(3) receptor (InsP(3)R) activity. This reduction was also found in peripheral neuronal tissue from Taxol treated animals. We further observed that short hairpin RNA-mediated NCS-1 knockdown had a similar effect on phosphoinositide-mediated Ca(2+) signaling. When NCS-1 protein levels recovered, so did InsP(3)-mediated Ca(2+) signaling. Inhibition of the Ca(2+)-activated protease mu-calpain prevented alterations in phosphoinositide-mediated Ca(2+) signaling and NCS-1 protein levels. We also found that NCS-1 is readily degraded by mu-calpain in vitro and that mu-calpain activity is increased in Taxol but not vehicle-treated cells. From these results, we conclude that prolonged exposure to Taxol activates mu-calpain, which leads to the degradation of NCS-1, which, in turn, attenuates InsP(3)mediated Ca(2+) signaling. These findings provide a previously undescribed approach to understanding and treating Taxol-induced peripheral neuropathy.

Nogo receptor antagonism promotes stroke recovery by enhancing axonal plasticity.

After ischemic stroke, partial recovery of function frequently occurs and may depend on the plasticity of axonal connections. Here, we examine whether blockade of the Nogo-NogoReceptor (NgR) pathway might enhance axonal sprouting and thereby recovery after focal brain infarction. Mutant mice lacking NgR or Nogo-AB recover complex motor function after stroke more completely than do control animals. After a stroke, greater numbers of axons emanating from the undamaged cortex cross the midline to innervate the contralateral red nucleus and the ipsilateral cervical spinal cord; this axonal plasticity is enhanced in ngr -/- or nogo-ab -/- mice. In rats with middle cerebral artery occlusion, both the recovery of motor skills and corticofugal axonal plasticity are promoted by intracerebroventricular administration of a function-blocking NgR fragment. Behavioral improvement occurs when therapy is initiated 1 week after arterial occlusion. Thus, delayed pharmacological blockade of the NgR promotes subacute stroke recovery by facilitating axonal plasticity.

A system for performing high throughput assays of synaptic function.

Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders.

TRPA1 mediates the noxious effects of natural sesquiterpene deterrents.

Plants, fungi, and animals generate a diverse array of deterrent natural products that induce avoidance behavior in biological adversaries. The largest known chemical family of deterrents are terpenes characterized by reactive alpha,beta-unsaturated dialdehyde moieties, including the drimane sesquiterpenes and other terpene species. Deterrent sesquiterpenes are potent activators of mammalian peripheral chemosensory neurons, causing pain and neurogenic inflammation. Despite their wide-spread synthesis and medicinal use as desensitizing analgesics, their molecular targets remain unknown. Here we show that isovelleral, a noxious fungal sesquiterpene, excites sensory neurons through activation of TPRA1, an ion channel involved in inflammatory pain signaling. TRPA1 is also activated by polygodial, a drimane sesquiterpene synthesized by plants and animals. TRPA1-deficient mice show greatly reduced nocifensive behavior in response to isovelleral, indicating that TRPA1 is the major receptor for deterrent sesquiterpenes in vivo. Isovelleral and polygodial represent the first fungal and animal small molecule agonists of nociceptive transient receptor potential channels.