Prostaglandin E2 EP1 receptors: downstream effectors of COX-2 neurotoxicity.

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid synthesis, has been implicated in the neurotoxicity resulting from hypoxia-ischemia, and its inhibition has therapeutic potential for ischemic stroke. However, COX-2 inhibitors increase the risk of cardiovascular complications. We therefore sought to identify the downstream effectors of COX-2 neurotoxicity, and found that prostaglandin E(2) EP1 receptors are essential for the neurotoxicity mediated by COX-2-derived prostaglandin E(2). EP1 receptors disrupt Ca(2+) homeostasis by impairing Na(+)-Ca(2+) exchange, a key mechanism by which neurons cope with excess Ca(2+) accumulation after an excitotoxic insult. Thus, EP1 receptors contribute to neurotoxicity by augmenting the Ca(2+) dysregulation underlying excitotoxic neuronal death. Pharmacological inhibition or gene inactivation of EP1 receptors ameliorates brain injury induced by excitotoxicity, oxygen glucose deprivation and middle cerebral artery (MCA) occlusion. An EP1 receptor inhibitor reduces brain injury when administered 6 hours after MCA occlusion, suggesting that EP1 receptor inhibition may be a viable therapeutic option in ischemic stroke.

Homer proteins regulate sensitivity to cocaine.

Drug addiction involves complex interactions between pharmacology and learning in genetically susceptible individuals. Members of the Homer gene family are regulated by acute and chronic cocaine administration. Here, we report that deletion of Homer1 or Homer2 in mice caused the same increase in sensitivity to cocaine-induced locomotion, conditioned reward, and augmented extracellular glutamate in nucleus accumbens as that elicited by withdrawal from repeated cocaine administration. Moreover, adeno-associated virus-mediated restoration of Homer2 in the accumbens of Homer2 KO mice reversed the cocaine-sensitized phenotype. Further analysis of Homer2 KO mice revealed extensive additional behavioral and neurochemical similarities to cocaine-sensitized animals, including accelerated acquisition of cocaine self-administration and altered regulation of glutamate by metabotropic glutamate receptors and cystine/glutamate exchange. These data show that Homer deletion mimics the behavioral and neurochemical phenotype produced by repeated cocaine administration and implicate Homer in regulating addiction to cocaine.

Inducible nitric oxide synthase contributes to gender differences in ischemic brain injury.

Estrogens have antiinflammatory actions and protect the brain from ischemic injury. Cerebral ischemia is accompanied by an inflammatory reaction that contributes to the tissue damage, an effect mediated in part by toxic amounts of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS). Therefore, estrogens may protect the female brain by modulating postischemic iNOS expression. To test this hypothesis, we studied whether iNOS plays a role in the mechanisms of the reduced susceptibility to ischemic injury observed in female mice. The middle cerebral artery was occluded for 20 mins using an intraluminal filament in C57Bl/6 mice, and infarct volume was assessed 3 days later in cresyl violet-stained sections. Infarcts were 53% smaller in female mice than in males (P < 0.05), a reduction abolished by ovariectomy (OVX) and reinstated by estrogen replacement. In normal female mice, postischemic iNOS mRNA was lower than in males (P < 0.05). Ovariectomy increased iNOS mRNA after ischemia and estrogen replacement blocked this effect. Furthermore, the iNOS inhibitor aminoguanidine reduced infarct volume in male, but not in female, mice. Similarly, male iNOS-null mice had smaller infarcts than wild-type mice, but female iNOS nulls were not protected. Ovariectomy and OVX with estrogen replacement did not affect infarct volume in iNOS-null female mice. The findings suggest that the neuroprotection conferred by estrogens is, in part, related to attenuation of iNOS expression. Such attenuation could result from the potent antiinflammatory effects of estrogens that downregulate iNOS expression via transcriptional or posttranscriptional mechanisms.

Dissociable roles for the dorsal and median raphé in the facilitatory effect of 5-HT1A receptor stimulation upon cocaine-induced locomotion and sensitization.

A distinct role for serotonin transmission from the dorsal and median raphé nuclei (DRN and MRN, respectively) was identified in regulating the behavioral and neurochemical effects of acute and repeated cocaine administration. Serotonin 1A (5-hydroxytryptophan (5-HT)1A) receptors were stimulated by intraraphé microinjection of 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT; 5 or 10 microg) and behavior, as well as extracellular neurotransmitter content in the nucleus accumbens was measured. Pretreatment of the DRN with DPAT caused a sensitization-like potentiation of acute cocaine-induced motor activity and an elevation in extracellular dopamine and glutamate. In contrast, DPAT microinjection into the MRN did not alter acute cocaine-induced motor activity or extracellular levels of dopamine or glutamate. Acutely, DPAT microinjection into either raphé nucleus reduced the basal and acute cocaine-stimulated levels of extracellular serotonin. Pretreatment with DPAT before systemic cocaine administration was continued for 5 days, and 3 weeks after the last injection, all rats were administered a cocaine challenge injection. The sensitized behavioral and neurochemical response produced by repeated cocaine in control subjects was unaffected by the intra-DRN administration of DPAT. However, in animals administered DPAT into the MRN, both the sensitized motor response and the increase in glutamate were augmented, while the sensitized serotonin response was blocked, without altering dopamine sensitization. These data show a differential role for 5-HT1A receptors in the DRN and MRN in the acute and sensitized effects of cocaine. While the DRN is involved in the acute effects of cocaine, neuroadaptations in the MRN may regulate the long-term consequences of repeated cocaine exposure.

Pretreatment with serotonin 5-HT(3) receptor antagonists produces no observable blockade of long-term motor sensitization to cocaine in rats.

RATIONALE: Serotonin 5-HT(3) receptor antagonists are proposed to serve as potential anti-addictive and anti-psychotic therapies. OBJECTIVE: The present study re-examined the hypothesis that the activation of 5-HT(3) receptors is required for the development of long-term motor sensitization to repeated cocaine (COC). METHODS: Rats were pretreated with vehicle (VEH) or one of three 5-HT(3) receptor antagonists, MDL 72222, Y-25130 or ondansetron, 30 min prior to seven daily COC or saline (SAL) injections. Three weeks later, animals were challenged with COC in a test for sensitization. For comparison, the effects of pretreatment with the dopamine (DA) receptor antagonist fluphenazine and a combination of fluphenazine and Y-25130 were assessed. RESULTS: Pretreatment with ondansetron, Y-25130, fluphenazine and their combination significantly attenuated COC-induced behaviors during repeated treatment but not on the test for sensitization. MDL 72222 pretreatment enhanced motor sensitization on the test day. In repeated SAL rats, pretreatment with ondansetron, Y-25130 and fluphenazine+Y-25130 enhanced COC-induced rearing on the test for sensitization. CONCLUSIONS: These data indicate the effects of 5-HT(3) receptor antagonists on both acute COC-induced motor behavior and COC-induced motor sensitization are compound-selective. As none of the 5-HT(3) receptor antagonists attenuated the magnitude of the sensitized motor response to COC in the long term, these data also indicate that like DA receptor activation, 5-HT(3) receptor activation is necessary for the full expression of acute COC-induced motor hyperactivity, but it is not required for the development of long-term motor sensitization.

Unconditioned and conditioned factors contribute to the 'reinstatement' of cocaine place conditioning following extinction in C57BL/6 mice.

Relapse to drug use following prolonged periods of abstinence results, in part, from the ability of contextual cues paired previously with self-administered drug to elicit drug craving and -seeking behavior. Given the popularity of the mouse for the genetic analysis of drug-induced behaviors, a place conditioning model of drug-seeking behavior was used to examine the ability of cocaine (COC) to reinstate extinguished conditioned reward in mice. In a series of experiments, COC place conditioning was produced in male C57BL/6 (B6) mice by four pairings of COC (15 or 25 mg/kg, IP) with the non-preferred compartment of a two-compartment place conditioning apparatus. Following a post-conditioning test (Post-Test), place conditioning was extinguished by repeated testing. The mice were then challenged with one of five COC doses (0, 5, 10, 15 or 25 mg/kg, IP) and allowed free access to both environments. Following extinction, COC injections reinstated place conditioning to 100% or greater, relative to the Post-Test. In a control experiment, mice received either COC or SAL paired with non-preferred compartment and were then challenged with either COC (15 mg/kg, IP) or SAL on the Post-Test. COC-conditioned, but not SAL-conditioned, mice exhibited place conditioning when tested in a COC-free state. Interestingly, COC injection on the Post-Test elicited an increase in approach behavior in both SAL- and COC-conditioned mice and this increase was equivalent to that produced by COC conditioning alone. No direct relationships were observed between the magnitude of place conditioning and either COC-induced or -conditioned locomotor hyperactivity in the non-preferred compartment. Thus, at least two independent processes appear to underlie the ability of a COC injection to elicit approach behavior towards the non-preferred compartment of a biased place conditioning apparatus in mice-reactivation of the conditioned incentive motivational properties of COC-paired cues and elicitation of unconditioned behavioral disinhibition. One or both of these processes sensitizes with the passage of time, increasing the propensity of B6 mice to approach non-preferred environments upon COC re-administration.

Changes in the subcellular distribution of NADPH oxidase subunit p47phox in dendrites of rat dorsomedial nucleus tractus solitarius neurons in response to chronic administration of hypertensive agents.

NADPH oxidase-generated superoxide can modulate crucial intracellular signaling cascades in neurons of the nucleus tractus solitarius (NTS), a brain region that plays an important role in cardiovascular processes. Modulation of NTS signaling by superoxide may be linked to the subcellular location of the mobile NADPH oxidase p47(phox) subunit, which is known to be present in dendrites of NTS neurons. It is not known, however, if hypertension can produce changes in the trafficking of p47(phox) in defined NTS subregions, particularly the preferentially barosensitive dorsomedial NTS (dmNTS), or preferentially gastrointestinal medial NTS (mNTS). We used immunogold electron microscopy to determine if p47(phox) localization was differentially affected in dendritic profiles of neurons from these NTS subregions of the rat in response to distinct models of hypertension, namely chronic 7-day subcutaneous administration of angiotensin II (AngII), or phenylephrine. In small (<1 microm) dendritic processes, both AngII and phenylephrine produced a decrease in intracellular p47(phox) labeling selectively in dmNTS neurons. In intermediate-size (1-2 microm) dendritic profiles in the dmNTS region only, there was an increase in p47(phox) labeling in response to each hypertensive agent, although these changes occurred in different subcellular compartments. There was an increase in non-vesicular labeling in response to AngII, but an increase in surface labeling with phenylephrine. Moreover, each of the changes in p47(phox) targeting mentioned above occurred in dendritic profiles with, or without immunoperoxidase labeling for the AngII AT-1A receptor subtype (AT-1A). These results indicate that chronic administration of agents that induce hypertension can also produce changes in the subcellular localization in p47(phox) in dmNTS neurons. Thus, systemic hypertension may produce alterations in the trafficking of proteins associated with superoxide production in central autonomic neurons, thus revealing a potentially important neurogenic component of free radical production and systemic blood pressure elevation.

Nox2, Ca2+, and protein kinase C play a role in angiotensin II-induced free radical production in nucleus tractus solitarius.

The dorsomedial portion of the nucleus tractus solitarius (dmNTS) is the site of termination of baroreceptor and cardiorespiratory vagal afferents and plays a critical role in cardiovascular regulation. Angiotensin II (Ang II) is a powerful signaling molecule in dmNTS neurons and exerts some of its biological effects by modulating Ca(2+) currents via reactive oxygen species (ROS) derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. We investigated whether a Nox2-containing NADPH oxidase is the source of the Ang II-induced ROS production and whether the signaling mechanisms of its activation require intracellular Ca(2+) or protein kinase C (PKC). Second-order dmNTS neurons were anterogradely labeled with 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide transported from the vagus and isolated from the brain stem. ROS production was assessed in 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide-positive dmNTS neurons using the fluorescent dye 6-carboxy-2',7'-dichlorodihydro-fluorescein di(acetoxymethyl ester). Ang II (3 to 2000 nmol/L) increased ROS production in dmNTS neurons (EC(50)=38.3 nmol/L). The effect was abolished by the ROS scavenger Mn (III) porphyrin 5,10,20-tetrakis (benzoic acid) porphyrin manganese (III), the Ang II type 1 receptor antagonist losartan, or the NADPH oxidase inhibitors apocynin or gp91ds-tat. Ang II failed to increase ROS production or to potentiate L-type Ca(2+) currents in dmNTS neurons of mice lacking Nox2. The PKC inhibitor GF109203X or depletion of intracellular Ca(2+) attenuated Ang II-elicited ROS production. We conclude that the powerful effects of Ang II on Ca(2+) currents in dmNTS neurons are mediated by PKC activation leading to ROS production via Nox2. Thus, a Nox2-containing NADPH oxidase is the critical link between Ang II and the enhancement of Ca(2+) currents that underlie the actions of Ang II on central autonomic regulation.