Activation Of NMDA Glutamate Receptors Research Articles

NMDA Receptors in the Lateral Preoptic Hypothalamus Are Essential for Sustaining NREM and REM Sleep.

The lateral preoptic (LPO) hypothalamus is a center for NREM and REM sleep induction and NREM sleep homeostasis. Although LPO is needed for NREM sleep, we found that calcium signals were, surprisingly, highest in REM sleep. Furthermore, and equally surprising, NMDA receptors in LPO were the main drivers of excitation. Deleting the NMDA receptor GluN1 subunit from LPO abolished calcium signals in all cells and produced insomnia. Mice of both sexes had highly fragmented NREM sleep-wake patterns and could not generate conventionally classified REM sleep. The sleep phenotype produced by deleting NMDA receptors depended on where in the hypothalamus the receptors were deleted. Deleting receptors from the anterior hypothalamic area did not influence sleep-wake states. The sleep fragmentation originated from NMDA receptors on GABA neurons in LPO. Sleep fragmentation could be transiently overcome with sleeping medication (zolpidem) or sedatives (dexmedetomidine). By contrast, fragmentation persisted under high sleep pressure produced by sleep deprivation - mice had a high propensity to sleep but woke up. By analyzing changes in delta power, sleep homeostasis (also referred to as “sleep drive”) remained intact after NMDA receptor ablation. We suggest NMDA glutamate receptor activation stabilizes firing of sleep-on neurons, and that mechanisms of sleep maintenance differ from that of the sleep drive itself.

Mitochondrial Carrier Homolog 2 Functionally Co-operates With BH3 Interacting-Domain Death Agonist in Promoting Ca2+-Induced Neuronal Injury.

The BH3 interacting-domain death agonist (BID) is a pro-apoptotic member of the Bcl-2 protein family. While proteolytic processing of BID links death receptor-induced apoptosis to the mitochondrial apoptosis pathway, we previously showed that full length BID also translocates to mitochondria during Ca2+-induced neuronal cell death. Moreover, mitochondrial carrier homolog 2 (MTCH2) was identified as a mitochondrial protein that interacts with BID during cell death. We started our studies by investigating the effect of Mtch2 silencing in a well-established model of Ca2+-induced mitochondrial permeability transition pore opening in non-neuronal HCT116 cells. We found that silencing of Mtch2 inhibited mitochondrial swelling and the associated decrease in mitochondrial energetics, suggesting a pro-death function for MTCH2 during Ca2+-induced injury. Next, we explored the role of BID and MTCH2 in mediating Ca2+-induced injury in primary cortical neurons triggered by prolonged activation of NMDA glutamate receptors. Analysis of intracellular Ca2+ transients, using time-lapse confocal microscopy, revealed that neurons lacking Bid showed markedly reduced Ca2+ levels during the NMDA excitation period. These Ca2+ transients were further decreased when Mtch2 was also silenced. Collectively, our data suggest that BID and MTCH2 functionally interact to promote Ca2+-induced neuronal injury.

α2δ-1 Upregulation in Primary Sensory Neurons Promotes NMDA Receptor-Mediated Glutamatergic Input in Resiniferatoxin-Induced Neuropathy.

Systemic treatment with resiniferatoxin (RTX) induces small-fiber sensory neuropathy by damaging TRPV1-expressing primary sensory neurons and causes distinct thermal sensory impairment and tactile allodynia, which resemble the unique clinical features of postherpetic neuralgia. However, the synaptic plasticity associated with RTX-induced tactile allodynia remains unknown. In this study, we found that RTX-induced neuropathy is associated with α2δ-1 upregulation in the dorsal root ganglion (DRG) and increased physical interaction between α2δ-1 and GluN1 in the spinal cord synaptosomes. RNAscope in situ hybridization showed that RTX treatment significantly increased α2δ-1 expression in DRG neurons labeled with calcitonin gene-related peptide, isolectin B4, NF200, and tyrosine hydroxylase. Electrophysiological recordings revealed that RTX treatment augmented the frequency of miniature excitatory postsynaptic currents (mEPSCs) and the amplitude of evoked EPSCs in spinal dorsal horn neurons, and these effects were reversed by blocking NMDA receptors with AP-5. Inhibiting α2δ-1 with gabapentin, genetically ablating α2δ-1, or targeting α2δ-1-bound NMDA receptors with α2δ-1Tat peptide largely normalized the baseline frequency of mEPSCs and the amplitude of evoked EPSCs potentiated by RTX treatment. Furthermore, systemic treatment with memantine or gabapentin and intrathecal injection of AP-5 or Tat-fused α2δ-1 C terminus peptide reversed allodynia in RTX-treated rats and mice. In addition, RTX-induced tactile allodynia was attenuated in α2δ-1 knock-out mice and in mice in which GluN1 was conditionally knocked out in DRG neurons. Collectively, our findings indicate that α2δ-1-bound NMDA receptors at presynaptic terminals of sprouting myelinated afferent nerves contribute to RTX-induced potentiation of nociceptive input to the spinal cord and tactile allodynia.SIGNIFICANCE STATEMENT Postherpetic neuralgia (PHN), associated with shingles, is a distinct form of neuropathic pain commonly seen in elderly and immunocompromised patients. The synaptic plasticity underlying touch-induced pain hypersensitivity in PHN remains unclear. Using a nonviral animal model of PHN, we found that glutamatergic input from primary sensory nerves to the spinal cord is increased via tonic activation of glutamate NMDA receptors. Also, we showed that α2δ-1 (encoded by Cacna2d1), originally considered a calcium channel subunit, serves as an auxiliary protein that promotes activation of presynaptic NMDA receptors and pain hypersensitivity. This new information advances our understanding of the molecular mechanism underlying PHN and suggests new strategies for treating this painful condition.

Purinergic Receptors in Basal Ganglia Diseases: Shared Molecular Mechanisms between Huntington's and Parkinson's Disease.

Huntington's (HD) and Parkinson's diseases (PD) are neurodegenerative disorders caused by the death of GABAergic and dopaminergic neurons in the basal ganglia leading to hyperkinetic and hypokinetic symptoms, respectively. We review here the participation of purinergic receptors through intracellular Ca2+ signaling in these neurodegenerative diseases. The adenosine A2A receptor stimulates striatopallidal GABAergic neurons, resulting in inhibitory actions on GABAergic neurons of the globus pallidus. A2A and dopamine D2 receptors form functional heteromeric complexes inducing allosteric inhibition, and A2A receptor activation results in motor inhibition. Furthermore, the A2A receptor physically and functionally interacts with glutamate receptors, mainly with the mGlu5 receptor subtype. This interaction facilitates glutamate release, resulting in NMDA glutamate receptor activation and an increase of Ca2+ influx. P2X7 receptor activation also promotes glutamate release and neuronal damage. Thus, modulation of purinergic receptor activity, such as A2A and P2X7 receptors, and subsequent aberrant Ca2+ signaling, might present interesting therapeutic potential for HD and PD.

Mitogen-activated protein kinase signaling mediates opioid-induced presynaptic NMDA receptor activation and analgesic tolerance.

Opioid-induced hyperalgesia and analgesic tolerance can lead to dose escalation and inadequate pain treatment with μ-opioid receptor agonists. Opioids cause tonic activation of glutamate NMDA receptors (NMDARs) at primary afferent terminals, increasing nociceptive input. However, the signaling mechanisms responsible for opioid-induced activation of pre-synaptic NMDARs in the spinal dorsal horn remain unclear. In this study, we determined the role of MAPK signaling in opioid-induced pre-synaptic NMDAR activation caused by chronic morphine administration. Whole-cell recordings of excitatory post-synaptic currents (EPSCs) were performed on dorsal horn neurons in rat spinal cord slices. Chronic morphine administration markedly increased the frequency of miniature EPSCs, increased the amplitude of monosynaptic EPSCs evoked from the dorsal root, and reduced the paired-pulse ratio of evoked EPSCs. These changes were fully reversed by an NMDAR antagonist and normalized by inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2), p38, or c-Jun N-terminal kinase (JNK). Furthermore, intrathecal injection of a selective ERK1/2, p38, or JNK inhibitor blocked pain hypersensitivity induced by chronic morphine treatment. These inhibitors also similarly attenuated a reduction in morphine's analgesic effect in rats. In addition, co-immunoprecipitation assays revealed that NMDARs formed a protein complex with ERK1/2, p38, and JNK in the spinal cord and that chronic morphine treatment increased physical interactions of NMDARs with these three MAPKs. Our findings suggest that opioid-induced hyperalgesia and analgesic tolerance are mediated by tonic activation of pre-synaptic NMDARs via three functionally interrelated MAPKs at the spinal cord level. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

HIE and neuroprotection

The neonatal brain is sensitive to hypoxic-ischemic insults that cause selective patterns on damage depending on the type and severity of the insult. An insult that damages the brain is associated with a neonatal encephalopathy that is associated with seizures. This encephalopathy reflects a rise in extracellular glutamate in the brain due to impaired glial pumping of neurotransmitter out of the synapse, which in turn triggers activation of NMDA glutamate receptors. Activation of NMDA receptors causes calcium to flow through the NMDA receptor channel, leading to activation of nNOS and production of nitric oxide (NO). High levels of NO combined with activation of apoptosis inducing factor (AIF) cause dysfunction of mitochondria, leading to a mixture of delayed apoptosis and necrosis. Mild hypothermia continued for three days in a neonatal intensive care unit has become the standard of care in most high income countries, and it is effective if applied soon enough after the insult. However prolonged insults or those remote from the time of delivery do not respond as well and other adjuvant therapies are needed to combine with hypothermia. Erythropoietin is one of these therapies, and another is carbon nanoparticles called dendrimers that can be administered intravenously and can carry drugs through the blood brain barrier and are taken up by activated microglia in the brain. Dendrimers carrying the anti-oxidant N-acetyl-cysteine (NAC) are neuroprotective in models of endotoxin induced brain injury. Follow-up studies demonstrate to efficacy of these strategies on motor and cognitive function in older children.

Bed nucleus of the stria terminalis NMDA receptors and nitric oxide modulate contextual fear conditioning in rats

The bed nucleus of the stria terminalis (BNST) modulates anxiety-like responses, including conditioned emotional responses. Evidence suggests that glutamatergic neurotransmission in the BNST plays a role in the modulation of defensive responses. However, little is known about the involvement of glutamate NMDA receptor activation within the BNST, and its resultant increase in nitric oxide (NO) levels, in the expression of contextual fear conditioning (CFC). We investigated whether the antagonism of NMDA receptors or the reduction of NO levels in the BNST would attenuate behavioral and autonomic responses (i.e. increase in arterial pressure and heart rate, and decrease in tail cutaneous temperature) of rats submitted to a CFC paradigm. Intra-BNST infusion of AP7, an NMDA receptor antagonist, attenuated both behavioral and autonomic changes induced by CFC. Similar results were observed with NPLA and c-PTIO, an nNOS inhibitor and an NO scavenger, respectively. A positive correlation between BNST NO levels and the time spent in freezing behavior was also observed for animals submitted to the CFC. These findings indicate that the expression of CFC involves a facilitation of BNST NMDA receptor-NO signaling.This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’.

Hippocampal Glutamate NMDA Receptor Loss Tracks Progression in Alzheimer’s Disease: Quantitative Autoradiography in Postmortem Human Brain

Early Alzheimer's disease (AD) is characterized by memory loss and hippocampal atrophy with relative sparing of basal ganglia. Activation of glutamate NMDA receptors in the hippocampus is an important step in memory formation. We measured the density of NMDA receptors in samples of hippocampus, entorhinal cortex and basal ganglia obtained from subjects who died with pathologically confirmed AD and age- and sex- matched non-demented controls. We found significant decreases in NMDA receptor density in the hippocampus and entorhinal cortex but not in the basal ganglia. Loss of NMDA receptors was significantly correlated with neuropathological progression as assessed by Braak staging postmortem. The same samples were probed for neuroinflammation by measuring the density and gene expression of translocator protein 18kDA (TSPO), an established marker of microglial activation. Unlike NMDA receptor loss, increased densities of TSPO were found in all of the brain regions sampled. However hippocampal, but not striatal TSPO density and gene expression were inversely correlated with NMDA receptor density and positively correlated with Braak stage, suggesting NMDA receptors exacerbate neuroniflammatory damage. The high correlation between hippocampal NMDA receptor loss and disease progression supports the use of non invasive imaging with NMDA receptor tracers and positron emission tomography as a superior method for diagnosis, staging and treatment monitoring of AD in vivo.

Stress-induced hyperalgesia is associated with a reduced and delayed GABA inhibitory control that enhances post-synaptic NMDA receptor activation in the spinal cord

GABA and glutamate are both affected by stress and are involved in nociception. Thus, we determined whether stress-induced enhancement of inflammatory hyperalgesia is mediated by an imbalance between glutamate and GABA neurotransmission. Male rats were subjected daily to 10 to 20minutes per day of either forced swimming (FS) or sham swimming for 3 consecutive days; nonconditioned rats served as controls. Some rats were treated i.p. with ketamine (5mg/kg), diazepam (2mg/kg), flumazenil (0.1mg/kg), or vehicle (0.9% NaCl), 30 to 60minutes before each conditioning session or nociception assessment. Pain behavior, spinal nociceptive neuronal activation and GABA and glutamate release were respectively evaluated by the formalin test, the expression of c-Fos and in vivo microdialysis of superficial laminae of the lumbar spinal cord, 48hours after the last conditioning session. Nitric oxide metabolites (NOx) were determined as markers of post-synaptic NMDA receptor activation. FS stress enhanced formalin-induced hyperalgesia, increased pain-elicited c-Fos expression, decreased basal and delayed pain-induced GABA release, and increased basal and induced glutamate release. Hyperalgesia and c-Fos overexpression were blocked only by prestress treatment with diazepam and post-stress treatment with ketamine, whereas changes in GABA and glutamate release were reversed by prestress treatment with diazepam. Diazepam effects were blocked by flumazenil. NOx increased in lumbar spinal cord of FS rats by a mechanism antagonized by ketamine. Thus, stress-induced hyperalgesia is initiated by a decreased and delayed GABA release and GABA-A receptor activation, whereas it is maintained by increased glutamate release and NMDA glutamate receptor activation at the spinal level.

Blocking the Deadly Effects of the NMDA Receptor in Stroke

Excessive activation of NMDA glutamate receptors contributes to neuronal death after stroke. In this issue, Tu et al. (2010) demonstrate that ischemic injury promotes the association of death-associated protein kinase 1 with the NMDA receptor, thereby potentiating its activity, and show that disrupting this association reduces damage to the brain.

The effect of ketamine and nitrous oxide on the human pupillary light reflex during general anesthesia

The neurotransmitters and receptor types involved in the afferent arm of the human pupillary light reflex are unknown. We hypothesized that the pupillary light reflex is mediated in part by NMDA receptors and that it would be depressed by the NMDA antagonists, nitrous oxide and ketamine. To study this question, sixteen patients received general anesthesia with desflurane, fentanyl, and muscular relaxation with rocuronium. After a stable level of general anesthesia had been obtained and at least 1 h after the start of the surgical procedure, ketamine 1 mg/kg ( N = 8) or saline ( N = 8) was injected intravenously by random selection. Heart rate, pupil size, pupillary light reflex, BIS scores, and blood pressure were measured every 2 min before and for 30 min after drug administration. A similar study of sixteen patients was then conducted with either addition of 60% nitrous oxide or 60% nitrogen to the gas mixture. We observed that the pupillary light reflex was depressed by ketamine and nitrous oxide by approximately 50%. The BIS score, representing the processed electroencephalogram, was elevated by ketamine and unchanged with nitrous oxide. Heart rate, pupil size, and blood pressure were unchanged by the drugs when compared to the control groups. We conclude that the two NMDA antagonists ketamine and nitrous oxide depress the human pupillary light reflex during general anesthesia whereas other monitored parameters were either unchanged or paradoxically elevated by the drugs. These findings present evidence that glutamate NMDA receptor activation is involved in generating the human pupillary light reflex.

The role of neonatal NMDA receptor activation in defeminization and masculinization of sex behavior in the rat

Normal development of the male rat brain involves two distinct processes, masculinization and defeminization, that occur during a critical period of brain sexual differentiation. Masculinization allows for the capacity to express male sex behavior in adulthood, and defeminization eliminates or suppresses the capacity to express female sex behavior in adulthood. Despite being separate processes, both masculinization and defeminization are induced by neonatal estradiol exposure. Though the mechanisms underlying estradiol-mediated masculinization of behavior during development have been identified, the mechanisms underlying defeminization are still unknown. We sought to determine whether neonatal activation of glutamate NMDA receptors is a necessary component of estradiol-induced defeminization of behavior. We report here that antagonizing glutamate receptors during the critical period of sexual differentiation blocks estradiol-induced defeminization but not masculinization of behavior in adulthood. However, enhancing NMDA receptor activation during the same critical period mimics estradiol to permanently induce both defeminization and masculinization of sexual behavior.

Quinolinate-induced Rat Striatal Excitotoxicity Impairs Endoplasmic Reticulum Ca2+-ATPase Function

Excessive activation of NMDA glutamate receptors and the resulting loss of intracellular Ca(2+) homeostasis may be lethal (excitotoxic) to neurons. Such excitotoxicity can be induced in vivo by intrastriatal infusion of quinolinate, as this substance selectively activates NMDA receptors. The aim of the present research was to investigate whether the in vivo treatment of striatal tissue with quinolinate would lead to an early impairment of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) activity or mitochondrial Ca(2+) sequestration, two intracellular mechanisms involved in Ca(2+) homeostasis and signaling. Sodium quinolinate was infused intrastriatally into adult rats, and 6 h later the brains were removed and the corpora striata dissected. At this time point, striatal sections stained with Fluoro-Jade, a cellular marker of cell death, showed initial signs of neuronal degeneration. In addition, SERCA activity decreased 39% in relation to the activity observed in the control striata. A corresponding decrease of the same magnitude in (45)Ca(2+) uptake by striatal microsomes was also found in the treated striata. Western blot analysis did not indicate any decrease in SERCA levels in striatal tissue after quinolinate infusion. Mitochondrial Ca(2+) sequestration was still preserved in quinolinate-treated striatal tissue when the assay was carried out in the presence of physiological concentrations of ATP and Mg(2+). These results suggest that impairment of the SERCA function may be an early event in excitotoxicity.

NMDA receptor blockade in the basolateral amygdala disrupts consolidation of stimulus-reward memory and extinction learning during reinstatement of cocaine-seeking in an animal model of relapse

Previous research from our laboratory has implicated the basolateral amygdala (BLA) complex in the acquisition and consolidation of cue–cocaine associations, as well as extinction learning, which may regulate the long-lasting control of conditioned stimuli (CS) over drug-seeking behavior. Given the well established role of NMDA glutamate receptor activation in other forms of amygdalar-based learning, we predicted that BLA-mediated drug–cue associative learning would be NMDA receptor dependent. To test this hypothesis, male Sprague–Dawley rats self-administered i.v. cocaine (0.6 mg/kg/infusion) in the absence of explicit CS pairings (2-h sessions, 5 days), followed by a single 1-h classical conditioning (CC) session, during which they received passive infusions of cocaine discretely paired with a light + tone stimulus complex. Following additional cocaine self-administration sessions in the absence of the CS (2-h sessions, 5 days) and extinction training sessions (no cocaine or CS presentation, 2-h sessions, 7 days), the ability of the CS to reinstate cocaine-seeking on three test days was assessed. Rats received bilateral intra-BLA infusions (0.5 μl/hemisphere) of vehicle or the selective NMDA receptor antagonist, 2-amino-5-phosphonovalerate (AP-5), immediately prior to the CC session (acquisition), immediately following the CC session (consolidation), or immediately following reinstatement testing (consolidation of conditioned-cued extinction learning). AP-5 administered before or after CC attenuated subsequent CS-induced reinstatement, whereas AP-5 administered immediately following the first two reinstatement tests impaired the extinction of cocaine-seeking behavior. These results suggest that NMDA receptor-mediated mechanisms within the BLA play a crucial role in the consolidation of drug–CS associations into long-term memories that, in turn, drive cocaine-seeking during relapse.

The Environment versus Genetics in Controlling the Contribution of MAP Kinases to Synaptic Plasticity

A challenge in biomedical research is to design experimental paradigms that reflect a natural setting. Even when freshly isolated tissues are used, they are almost always derived from animals housed in cages that poorly reflect the animal's native environment. This issue is highlighted by studies on brain function, where mice housed in a more natural "enriched environment" display enhanced learning and memory and delayed onset of symptoms of neurodegenerative diseases compared to mice housed conventionally. How the environment mediates its effects on brain function is poorly understood. We show that after exposure of adolescent mice to an "enriched environment," the induction of long-term potentiation (LTP), a form of synaptic plasticity that is thought to contribute to learning and memory, involves a novel signal transduction pathway that is nonfunctional in comparable mice housed conventionally. This environmentally gated signaling pathway, which rescues defective LTP induction in adolescent Ras-GRF knockout mice, consists of NMDA glutamate receptor activation of p38, a MAP kinase that does not contribute to LTP in mice housed conventionally. Interestingly, the same exposure to environmental enrichment does not have this effect in adult mice. This study reveals a new level of cell signaling control whereby environmental factors gate the efficacy of a specific signaling cascade to control how LTP is induced in adolescent animals. The suppression of this gating mechanism in mature animals represents a new form of age-dependent decline in brain plasticity.

Getting his act together: Roles of glutamate, nitric oxide, and dopamine in the medial preoptic area

Gonadal hormones have primarily slow, genomically mediated effects, but copulation requires rapid interactions with a partner. A major way in which hormones facilitate male sexual behavior is by increasing production of neurotransmitter receptors or of enzymes that regulate neurotransmitter synthesis or release. Dopamine is an important facilitative neurotransmitter, and the medial preoptic area (MPOA) is a critical integrative site for male sexual behavior. MPOA dopamine is released before and during mating and facilitates copulation, genital reflexes, and sexual motivation. Gonadal hormones regulate dopamine release in the MPOA of male rats in part by increasing nitric oxide synthase (NOS) in the MPOA; the resultant increase in production of nitric oxide (NO) increases both basal and female-stimulated dopamine release. Glutamate also increases dopamine release via increased production of NO. At least some of the glutamatergic inputs to the MPOA are from the medial amygdala (MeA) and bed nucleus of the stria terminalis (BNST), which mediate the female-stimulated increase in dopamine, which in turn enhances copulatory ability. Extracellular glutamate in the MPOA increases during copulation, especially during ejaculation, and increased glutamate facilitates copulation and genital reflexes. Previous sexual experience also facilitates copulation and confers resistance to impairment by various lesions, drugs, and stress. Experience enhances processing of sexual stimuli, and its effects require activation of glutamate NMDA receptors and NOS in the MPOA. Neuronal NOS is increased in the MPOA of experienced males. Therefore, glutamate, NO, and dopamine interact in the MPOA to facilitate mating and to enhance future sexual responsiveness.

Testosterone induces neuroprotection from oxidative stress. Effects on catalase activity and 3-nitro-L-tyrosine incorporation into alpha-tubulin in a mouse neuroblastoma cell line.

3-nitro-L-tyrosine is formed by nitric oxide following different pathways such as NADPH oxidase, xanthine oxidase or glutamate NMDA receptor activation and is involved in the pathology of different neurological disorders. Unlike estradiol, a neuroprotective role of androgens against oxidative cell injury has not been fully investigated. This work targets the possible effects of testosterone on neuroblastoma cells exposed to 3-nitro-L-tyrosine. C1300 mouse undifferentiated neuroblastoma cells exposed to 3-nitro-L-tyrosine were cultured in the presence of testosterone. Morphological examination, proliferation and nuclear viability assays were performed. The expression of tyrosinated alpha-tubulin and incorporation of 3-nitro-L-tyrosine into protein were also estimated. Cells exposed to 3-nitro-L-tyrosine showed globular shape, reduced cytoplasmic processes and growth inhibition in comparison with controls. When testosterone was added to the medium, these changes were not evident. In addition, testosterone induced an upregulation of tyrosinated alpha-tubulin, a marker of neuronal plasticity, and a decrease in 3-nitro-L-tyrosine incorporation into tubulin. Our results suggest that testosterone exposure can diminish 3-nitro-L-tyrosine toxic effects on the morphology and growth rate of neuroblastoma cells. The upregulation of tyrosinated alpha-tubulin in testosterone-exposed cells would be consistent with concurrent plasticity events. Failure in alpha-tubulin nitration detected in cells exposed to both 3-nitro-L-tyrosine and testosterone, may support the idea that testosterone interferes with 3-nitro-L-tyrosine protein incorporation. Moreover, testosterone-induced neuroprotection likely entails a linkage with the androgen receptor as is suggested by the flutamide-induced inhibition of the hormone activity. Finally, the neuroprotective effects of testosterone in neuroblastoma cells could deal with the cellular antioxidant defence system, as shown by testosterone-induced increase in catalase activity.

C-Fos expression increase in NADPH-diaphorase positive neurons after exposure to a live cat

The present study investigated if NOS positive neurons localized in regions related to defensive reactions are activated after exposure to an innate fear stimulus (a live cat). Male Wistar rats were exposed to a live or a toy cat for 10 min and 2 h later had their brains removed and processed for c-Fos immunohistochemistry (a marker of neuronal functional activation) and NADPH-diaphorase (NADPH-d; used to detect the presence of NOS neurons) histochemistry. Cat exposure induced a small (11%) to moderate (50%) significant increase in the percentage of double-stained cells (c-Fos + NADPH-d positive neurons) in the anteromedial bed nucleus of stria terminalis (BSTMA), medial amygdala (MeA), parvocellular paraventricular (pPVN), lateral (LH) and dorsal premammillary (PMd) hypothalamic nuclei, dorsolateral periaqueductal grey (dlPAG) and dorsal raphe nucleus (DRN). This increase was attenuated in the PMd, DRN and dlPAG by i.c.v. injection of AP7 (5 nmol/2 μl), an NMDA receptor antagonist. The drug increased the percentage of time the rats remained close to the cat in the observation box. The results suggest that exposure to a live predator activates neurons containing NOS in brain areas related to defensive reactions. They also indicate that this effect probably involves activation of NMDA glutamate receptors.

Defensive-like behaviors and antinociception induced by NMDA injection into the periaqueductal gray of mice depend on nitric oxide synthesis

Glutamate NMDA receptor activation within the periaqueductal gray (PAG) leads to antinociceptive, autonomic and behavioral responses characterized as the fear reaction. Considering that NMDA receptor triggers activation of neuronal nitric oxide synthase (nNOS), enzyme that produces nitric oxide (NO), this study investigated the effects of intra-PAG infusions of NPLA ( Nω-propyl- l-arginine), an nNOS inhibitor, on behavioral and antinociceptive responses induced by local injection of NMDA receptor agonist in mice. The behaviors measured were frequency of jumping and rearing as well as duration (in seconds) of running and freezing. Nociception was assessed during the second phase of the formalin test (injection of 50 μl of formalin 2.5% into the dorsal surface of the right hind paw). Five to seven days after stereotaxic surgery for intracerebral cannula implantation, mice were injected with formalin into the paw, and 10 min later, they received intra-dPAG injection of NPLA (0, 0.2, or 0.4 nmol/0.1 μl). Ten minutes later, they were injected with NMDA ( N-methyl- d-aspartate: 0 or 0.04 nmol/0.1 μl) into the same midbrain site and were immediately placed in glass holding cage for recording the defensive behavior and the time spent on licking the injected paw with formalin during a period of 10 min. Microinjections of NMDA significantly decreased nociception response and produced jumping, running, and freezing reactions. Intra-dPAG injections of NPLA (0.4 nmol) completely blocked the NMDA effects without affecting either behavioral or nociceptive responses in intra-dPAG saline-injected animals, except for the rearing frequency that was increased by the nNOS inhibitor. These results strongly suggest the involvement of NO within the PAG in the antinociceptive and defensive reactions induced by local glutamate NMDA receptor activation in this midbrain structure.

Forebrain Overexpression of CaMKII abolishes Cingulate Long Term Depression and Reduces Mechanical Allodynia and Thermal Hyperalgesia

Activity-dependent synaptic plasticity is known to be important in learning and memory, persistent pain and drug addiction. Glutamate NMDA receptor activation stimulates several protein kinases, which then trigger biochemical cascades that lead to modifications in synaptic efficacy. Genetic and pharmacological techniques have been used to show a role for Ca2+/calmodulin-dependent kinase II (CaMKII) in synaptic plasticity and memory formation. However, it is not known if increasing CaMKII activity in forebrain areas affects behavioral responses to tissue injury. Using genetic and pharmacological techniques, we were able to temporally and spatially restrict the over expression of CaMKII in forebrain areas. Here we show that genetic overexpression of CaMKII in the mouse forebrain selectively inhibits tissue injury-induced behavioral sensitization, including allodynia and hyperalgesia, while behavioral responses to acute noxious stimuli remain intact. CaMKII overexpression also inhibited synaptic depression induced by a prolonged repetitive stimulation in the ACC, suggesting an important role for CaMKII in the regulation of cingulate neurons. Our results suggest that neuronal CaMKII activity in the forebrain plays a role in persistent pain.