Network Asynchrony Underlying Increased Broadband Gamma Power.

Synchronous activity of cortical inhibitory interneurons expressing parvalbumin (PV) underlies expression of cortical γ rhythms. Paradoxically, deficient PV inhibition is associated with increased broadband γ power in the local field potential. Increased baseline broadband γ is also a prominent characteristic in schizophrenia and a hallmark of network alterations induced by NMDAR antagonists, such as ketamine. Whether enhanced broadband γ is a true rhythm, and if so, whether rhythmic PV inhibition is involved or not, is debated. Asynchronous and increased firing activities are thought to contribute to broadband power increases spanning the γ band. Using male and female mice lacking NMDAR activity specifically in PV neurons to model deficient PV inhibition, we here show that neuronal activity with decreased synchronicity is associated with increased prefrontal broadband γ power. Specifically, reduced spike time precision and spectral leakage of spiking activity because of higher firing rates (spike "contamination") affect the broadband γ band. Desynchronization was evident at multiple time scales, with reduced spike entrainment to the local field potential, reduced cross-frequency coupling, and fragmentation of brain states. Local application of S(+)-ketamine in (control) mice with intact NMDAR activity in PV neurons triggered network desynchronization and enhanced broadband γ power. However, our investigations suggest that disparate mechanisms underlie increased broadband γ power caused by genetic alteration of PV interneurons and ketamine-induced power increases in broadband γ. Our study confirms that enhanced broadband γ power can arise from asynchronous activities and demonstrates that long-term deficiency of PV inhibition can be a contributor.SIGNIFICANCE STATEMENT Brain oscillations are fundamental to the coordination of neuronal activity across neurons and structures. γ oscillations (30-80 Hz) have received particular attention through their association with perceptual and cognitive processes. Synchronous activity of inhibitory parvalbumin (PV) interneurons generates cortical γ oscillation, but, paradoxically, PV neuron deficiency is associated with increases in γ oscillations. We here reconcile this conundrum and show how deficient PV inhibition can lead to increased and asynchronous excitatory firing, contaminating the local field potential and manifesting as increased γ power. Thus, increased γ power does not always reflect a genuine rhythm. Further, we show that ketamine-induced γ increases are caused by separate network mechanisms.

Urokinase-type plasminogen activator (uPA) regulates the expression and function of growth-associated protein 43 (GAP-43) in the synapse.

Growth-associated protein 43 (GAP-43) plays a central role in the formation of presynaptic terminals, synaptic plasticity, and axonal growth and regeneration. During development, GAP-43 is found in axonal extensions of most neurons. In contrast, in the mature brain, its expression is restricted to a few presynaptic terminals and scattered axonal growth cones. Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin and activates signaling pathways that promote cell migration, proliferation, and survival. In the developing brain, uPA induces neuritogenesis and neuronal migration. In contrast, the expression and function of uPA in the mature brain are poorly understood. However, recent evidence reveals that different forms of injury induce release of uPA and expression of uPAR in neurons and that uPA/uPAR binding triggers axonal growth and synapse formation. Here we show that binding of uPA to uPAR induces not only the mobilization of GAP-43 from the axonal shaft to the presynaptic terminal but also its activation in the axonal bouton by PKC-induced calcium-dependent phosphorylation at Ser-41 (pGAP-43). We found that this effect requires open presynaptic N-methyl-d-aspartate receptors but not plasmin generation. Furthermore, our work reveals that, following its activation by uPA/uPAR binding, pGAP-43 colocalizes with presynaptic vesicles and triggers their mobilization to the synaptic release site. Together, these data reveal a novel role of uPA as an activator of the synaptic vesicle cycle in cerebral cortical neurons via its ability to induce presynaptic recruitment and activation of GAP-43.

GluN2A- and GluN2B-immunoreactive type I cells attached to vesicular glutamate transporter 2-immunoreactive afferent nerve terminals of the rat carotid body.

We previously reported the immunoreactivity for the vesicular glutamate transporter 2 (VGLUT2) in afferent nerve terminals attached to chemoreceptor type I cells of the carotid body (CB), suggesting that glutamate is released from afferent terminals to stimulate these cells. In the present study, we examined the immunoreactivity for the glutamate-binding subunits of N-methyl-D-aspartate (NMDA) receptors, GluN2A and GluN2B in the rat CB, and the immunohistochemical relationships between these subunits and VGLUT2. Immunoreactivities for GluN2A and GluN2B were predominant in a subpopulation of tyrosine hydroxylase-immunoreactive type I cells rather than those of dopamine beta-hydroxylase-immunoreactive cells. Punctate VGLUT2-immunoreactive products were attached to GluN2A- and GluN2B-immunoreactive type I cells. Bassoon-immunoreactive products were localized between VGLUT2-immunoreactive puncta and type I cells immunoreactive for GluN2A and GluN2B. These results suggest that afferent nerve terminals release glutamate by exocytosis to modulate chemosensory activity of a subpopulation of type I cells via GluN2A- and GluN2B subunits-containing NMDA receptors.

An Effective Phytoconstituent Aconitine: A Realistic Approach for the Treatment of Trigeminal Neuralgia.

Trigeminal neuralgia pain remains a challenge to treat. Natural compounds may be promising options for relieving pain. This study was aimed at investigating the effects of aconitine in a rat model of trigeminal neuralgia pain. Infraorbital nerve chronic constriction injury was performed in adult Wistar Albino rats. After the neuropathic pain developed, the rats were assigned to one of the treatment groups: carbamazepine 40 or 80 mg/kg; aconitine 0.25, 0.50, or 0.75 mg/kg; or saline injection (control group). Behavioral testing with von Frey filaments and the rotarod test were carried out before the surgical procedure and on the 24th to 29th postoperative days. Following the completion of tests, ipsilateral and contralateral spinal cords were harvested for Western blot analyses to assess NR-1 protein expression. ANOVA followed by Mann-Whitney U test was performed for the statistical analyses. P values of <0.05 were considered significant. Aconitine significantly reduced mechanical sensitivity in a dose-dependent manner. A significant reduction in motor coordination was noted for the higher doses of aconitine which was similar with the 40 and 80 mg/kg doses of carbamazepine. NR-1 expression was reduced in the ipsilateral spinal cord, whereas no significant difference was noted between the groups in the expression of NR-1 in the contralateral spinal cord. Aconitine had a significant pain relieving effect, which was similar to carbamazepine, in a dose-dependent manner. Aconitine may be an alternative pharmacological agent for the control of trigeminal neuralgia pain.

Anesthesia plus surgery in neonatal period impairs preference for social novelty in mice at the juvenile age.

Anesthetic sevoflurane could induce neurotoxicity in developing brain and cause adverse neurobehavioral outcomes in mice, including inattention, social interaction deficit, and learning and memory impairment. However, there is less data on the effect of anesthesia plus surgery on social interaction behavior. Therefore, we investigated whether the combination of anesthesia and surgical stimulation could induce behavioral and biochemical changes in mice. Firstly, the six-day-old mice were received either 3% sevoflurane anesthesia or abdominal surgery under sevoflurane anesthesia. Then, these mice were scheduled to social interaction test in three-chambered social paradigm at one-month-old. In addition, the brain tissues of neonatal mice were harvested at 24 h after treatment, for measuring the levels of OXTR and NMDAR1 in Western blot analysis. We found that neonatal anesthesia with sevoflurane in a clinically-relevant dosage could not induce social interaction deficit. Nevertheless, anesthesia plus surgery was able to impair preference for social novelty in mice. Moreover, anesthesia plus surgery decreased the levels of OXTR in hippocampus and cortex of mice, as well as NMDAR1 in hippocampus. Collectively, these results suggested that anesthesia plus surgery could impair social novelty preference, but not sociability in mice, and that social memory might be more vulnerable than social affiliation in biological property. Furthermore, reduction in the levels of cortex OXTR and hippocampus NMDAR1 could be associated with social recognition memory in mice.

Less NMDA Receptor Binding in Dorsolateral Prefrontal Cortex and Anterior Cingulate Cortex Associated With Reported Early-Life Adversity but Not Suicide.

BACKGROUND: Glutamate is an excitatory neurotransmitter binding to 3 classes of receptors, including the N-methyl, D-aspartate (NMDA) receptor. NMDA receptor binding is lower in major depression disorder and suicide. NMDA receptor blocking with ketamine can have antidepressant and anti-suicide effects. Early-life adversity (ELA) may cause glutamate-mediated excitotoxicity and is more common with major depression disorder and in suicide decedents. We sought to determine whether NMDA-receptor binding is altered with suicide and ELA. METHODS: A total 52 postmortem cases were organized as 13 quadruplets of suicide and non-suicide decedents matched for age, sex, and postmortem interval, with or without reported ELA (≤16 years). Tissue blocks containing dorsal prefrontal (BA8), dorsolateral prefrontal (BA9), or anterior cingulate (BA24) cortex were collected at autopsy. Psychiatrically healthy controls and suicide decedents underwent psychological autopsy to determine psychiatric diagnoses and details of childhood adversity. NMDA receptor binding was determined by quantitative autoradiography of [3H]MK-801 binding (displaced by unlabeled MK-801) in 20-µm-thick sections. RESULTS: [3H]MK-801 binding was not associated with suicide in BA8, BA9, or BA24. However, [3H]MK-801 binding with ELA was less in BA8, BA9, and BA24 independent of suicide (P < .05). [3H]MK-801 binding was not associated with age or postmortem interval in any brain region or group. CONCLUSIONS: Less NMDA receptor binding with ELA is consistent with the hypothesis that stress can cause excitotoxicity via excessive glutamate, causing either NMDA receptor downregulation or less receptor binding due to neuron loss consequent to the excitotoxicity.

Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain.

Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function.

Neuroprotective Effect of Astragalus Polysacharin on Streptozotocin (STZ)-Induced Diabetic Rats.

BACKGROUND In the recent years, there has been increasing interest in traditional Chinese medicine as a neuroprotective nutrient in the management of chronic neurodegenerative disease, such as diabetic cognitive decline. Astragalus polysacharin (APS), a Chinese herb extract, is a biologically active treatment for neurodegenerative diseases. Therefore, in the present study, we investigated the neuroprotective effects of APS (20 mg/kg) on diabetes-induced memory impairments in Sprague-Dawley (SD) rats and explored its underlying mechanisms of action. MATERIAL AND METHODS Thirty SD rats were randomly divided into a control group (CON group, n=10), a diabetic model (DM) group (n=10), and an APS group (n=10). We administered 55 mg/kg streptozotocin (STZ, Sigma) by intraperitoneal injection to induce a diabetic model. Food and water intake, body weight, and blood fasting plasma glucose (FPG) were measured. The Morris water maze test (MWM) was used to assess learning and memory ability, and we measured levels of N-methyl-D-aspartate receptor (NMDA), calcium/calmodulin-dependent protein kinase II (CaMKII), and cAMP response element-binding protein (CREB) in the hippocampus. RESULTS APS (20 mg/kg) administration decreased the rats' fasting plasma glucose (FPG) levels and body weight. APS (20 mg/kg) administration improved the cognitive performance of diabetes-induced rats in the Morris water maze test. APS (20 mg/kg) administration reduced the number of dead cells in the CA1 region of the hippocampus. Furthermore, APS (20 mg/kg) administration obviously upregulated the phosphorylation levels CREB, NMDA, and CaMK II. CONCLUSIONS These results suggest that APS has the neuroprotective effects, and it may be a candidate for treatment of neurodegenerative diseases such as diabetic cognitive impairment.

Deciphering GRINA/Lifeguard1: Nuclear Location, Ca2+ Homeostasis and Vesicle Transport.

The Glutamate Receptor Ionotropic NMDA-Associated Protein 1 (GRINA) belongs to the Lifeguard family and is involved in calcium homeostasis, which governs key processes, such as cell survival or the release of neurotransmitters. GRINA is mainly associated with membranes of the endoplasmic reticulum, Golgi, endosome, and the cell surface, but its presence in the nucleus has not been explained yet. Here we dissect, with the help of different software tools, the potential roles of GRINA in the cell and how they may be altered in diseases, such as schizophrenia or celiac disease. We describe for the first time that the cytoplasmic N-terminal half of GRINA (which spans a Proline-rich domain) contains a potential DNA-binding sequence, in addition to cleavage target sites and probable PY-nuclear localization sequences, that may enable it to be released from the rest of the protein and enter the nucleus under suitable conditions, where it could participate in the transcription, alternative splicing, and mRNA export of a subset of genes likely involved in lipid and sterol synthesis, ribosome biogenesis, or cell cycle progression. To support these findings, we include additional evidence based on an exhaustive review of the literature and our preliminary data of the protein-protein interaction network of GRINA.

Serum complement levels in anti-N-methyl-d-aspartate receptor encephalitis.

BACKGROUND AND PURPOSE: This study aimed to evaluate the relationship between serum complement and anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis. METHODS: Serum complement (C3, C4 and CH50), immunoglobulins (IgG, IgM and IgA) and C-reactive protein (CRP) were evaluated in 40 patients with anti-NMDAR encephalitis and 40 controls. Follow-up evaluations of 11 of the 40 patients with anti-NMDAR encephalitis were conducted 6 months after admission. Modified Rankin Scale (mRS) scores and clinical and cerebrospinal fluid parameters were evaluated in patients with anti-NMDAR encephalitis. RESULTS: Serum C4 levels were significantly higher in patients with anti-NMDAR encephalitis than in controls (P = 0.003), especially in female patients (P = 0.001) and those with severe impairment (mRS ≥ 4; P < 0.001). Serum CH50 levels were significantly higher in patients with severe impairment (P = 0.007) and limited treatment responses (P = 0.007). Serum C4 was associated with C3 (r = 0.506, P = 0.001), CH50 (r = 0.478, P = 0.002) and mRS score (r = 0.607, P < 0.001). Serum C3 was associated with CH50 (r = 0.339, P = 0.032) and cerebrospinal fluid white blood cells (r = 0.351, P = 0.026). Serum CH50 was associated with age, mRS score and CRP. Follow-up evaluations revealed that mRS scores were significantly lower than those before treatment, and a significant negative correlation was observed between the change in C3 levels and the change in mRS score. CONCLUSION: Our results demonstrated that serum C4 levels were elevated and associated with C3, CH50 and CRP levels, and disease severity in patients with anti-NMDAR encephalitis.

Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging.

Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.

Geniposide Attenuates Post-Ischaemic Neurovascular Damage via GluN2A/AKT/ ERK-Dependent Mechanism.

BACKGROUND/AIMS: Calcium-permeable ionotropic NMDAR-mediated hyperactivity is regarded as the critical factor in modulating the development of ischaemic stroke. Recently, there has been increasing interest in preventing post-stroke neuronal death by focusing on intervening in the function of subpopulations of NMDARs and their downstream signalling. Geniposide, an iridoid glycoside, has been found to have cytoprotective functions in various conditions. However, it is still unclear whether and how geniposide affects neuronal insult under experimental stroke. METHODS: We demonstrate that dose-dependent geniposide significantly decreased the infarct volume in tMCAO models. RESULTS: A medium level of geniposide improved anti-apoptotic functions and inhibited BBB leakage/haemorrhage via elevating GluN2A-containing NMDAR expression in tMCAO rats. Importantly, these effects could be eliminated by co-treatment of geniposide with the GluN2A antagonist NVP but not the GluN2B inhibitor ifenprodil. Moreover, geniposide's protection was due to the enhancement of GluN2A-dependent survival signals, including pAKT, pERK and PSD-95. CONCLUSION: The results suggest that geniposide protects neurons against post-ischaemic neurovascular injury through the activation of GluN2A/AKT/ERK pathways. As a very promising natural agent, geniposide may be a future therapeutic for stroke patients.

Psychosis in the ED: A case of NMDA receptor antibody encephalitis.

Anti-N-methyl-d-aspartate antibody receptor (NMDAR) encephalitis is a newly recognized disease increasing in diagnostic frequency. A 27-year-old female presented with symptoms of oral dyskinesia, tachycardia, and altered mental status following a three-month history of depression, lethargy, catatonia, and auditory hallucinations. We utilized our facilities neurology and psychiatry consult services, performed a lumbar puncture (LP), and requested NMDAR antibody titers. Following admission the Anti-NMDAR antibody titer was elevated warranting treatment with intravenous immunoglobulin (IVIG), corticosteroids, and later rituximab. Organic causes of psychosis are often overlooked in the emergency department, particularly in patients with a history of psychiatric illness. An understanding and awareness of NMDAR encephalitis allows for timely diagnosis, prompting quicker treatment. Emergency physicians should maintain an index of clinical suspicion for NMDAR encephalitis when encountering patients with progressive symptoms of catatonia and psychosis of unclear etiology.

ANKS1B Gene Product AIDA-1 Controls Hippocampal Synaptic Transmission by Regulating GluN2B Subunit Localization.

NMDA receptors (NMDARs) are key mediators of glutamatergic transmission and synaptic plasticity, and their dysregulation has been linked to diverse neuropsychiatric and neurodegenerative disorders. While normal NMDAR function requires regulated expression and trafficking of its different subunits, the molecular mechanisms underlying these processes are not fully understood. Here we report that the amyloid precursor protein intracellular domain associated-1 protein (AIDA-1), which associates with NMDARs and is encoded by ANKS1B, a gene recently linked to schizophrenia, regulates synaptic NMDAR subunit composition. Forebrain-specific AIDA-1 conditional knock-out (cKO) mice exhibit reduced GluN2B-mediated and increased GluN2A-mediated synaptic transmission, and biochemical analyses show AIDA-1 cKO mice have low GluN2B and high GluN2A protein levels at isolated hippocampal synaptic junctions compared with controls. These results are corroborated by immunocytochemical and electrophysiological analyses in primary neuronal cultures following acute lentiviral shRNA-mediated knockdown of AIDA-1. Moreover, hippocampal NMDAR-dependent but not metabotropic glutamate receptor-dependent plasticity is impaired in AIDA-1 cKO mice, further supporting a role for AIDA-1 in synaptic NMDAR function. We also demonstrate that AIDA-1 preferentially associates with GluN2B and with the adaptor protein Ca(2+)/calmodulin-dependent serine protein kinase and kinesin KIF17, which regulate the transport of GluN2B-containing NMDARs from the endoplasmic reticulum (ER) to synapses. Consistent with this function, GluN2B accumulates in ER-enriched fractions in AIDA-1 cKO mice. These findings suggest that AIDA-1 regulates NMDAR subunit composition at synapses by facilitating transport of GluN2B from the ER to synapses, which is critical for NMDAR plasticity. Our work provides an explanation for how AIDA-1 dysfunction might contribute to neuropsychiatric conditions, such as schizophrenia.

Neonatal Ethanol Exposure Impairs Trace Fear Conditioning and Alters NMDA Receptor Subunit Expression in Adult Male and Female Rats.

BACKGROUND: Ethanol (EtOH) exposure in neonate rats during a period comparable to the human third trimester, postnatal days (PD) 4 to 9, leads to persistent deficits in forebrain-dependent cognitive function--modeling the dysfunction seen in individuals diagnosed with fetal alcohol spectrum disorders. EtOH-exposed adult rats are impaired in auditory trace fear conditioning (TFC), a form of Pavlovian conditioning in which a neutral conditioned stimulus (CS; tone) is followed by an aversive unconditioned stimulus (US; footshock), with both stimuli separated in time by a stimulus-free "trace" interval (TI). TFC acquisition depends on N-methyl-d-aspartate NMDA receptor (NMDAR) activation in the dorsal hippocampus (DH), ventral hippocampus (VH), and medial prefrontal cortex (mPFC). METHODS: Male and female rat pups were sham-intubated (SI) or intragastrically intubated with EtOH (5E; 5 g/kg/d) over PD 4 to 9 and, as adults, submitted to TFC with a 15-second tone CS and 30-second TI. Whole-cell tissue lysates from the DH, VH, and mPFC of TFC rats and DH synaptic/extrasynaptic membrane fractions from experimentally naïve animals were analyzed via Western blot for NMDAR subunit (GluN1, GluN2A, GluN2B) expression. RESULTS: Freezing behavior during CS-alone test trials was significantly reduced in both male and female 5E rats, relative to same-sex controls. Western blot results based on DH tissue samples revealed a greater proportion of GluN2A to GluN2B subunits in 5E rats, relative to SI rats, and significantly reduced synaptic GluN2B and PSD-95 expression. CONCLUSIONS: EtOH-induced changes in DH NMDAR subunit expression-particularly synaptic GluN2B, which is critical for TFC-are proposed to weaken long-term memory consolidation and, during behavioral testing, diminish CS-evoked freezing behavior.

Anti-N-Methyl-d-Aspartate Receptor Encephalitis as an Unusual Cause of Altered Mental Status in the Emergency Department.

BACKGROUND: Anti-N-methyl-d-aspartate (NMDA) receptor autoimmune encephalitis is a newly identified form of encephalitis whose incidence is on the rise. Awareness of this condition and symptom recognition are key to early diagnosis and prompt treatment, which may alter the course of the disease. CASE REPORT: A 35-year-old woman presented to our Emergency Department (ED) with lethargy, bizarre behavior, agitation, confusion, memory deficits, and word-finding difficulties. Her symptoms and evaluation were potentially consistent with a primary psychiatric disorder, but the absence of frank psychosis and presence of neurologic features related to memory and cognition prompted other considerations. In the ED we performed a lumbar puncture, and in addition to routine studies, ordered anti-NMDAR antibody screening. The screening studies returned positive, leading to treatment with glucocorticoids and intravenous immune globulin and resulting in improvement to near baseline function. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Although anti-NMDAR encephalitis is relatively uncommon, reports of this previously unrecognized condition are increasing, with an unclear true incidence of disease. Emergency providers should consider this diagnosis in their differential for patients presenting with new neuropsychiatric symptoms, particularly in young women. Prompt treatment leads to near complete neurologic recovery in 75% of patients, whereas delays in diagnosis and treatment may be associated with worse outcomes including death.

Increased susceptibility of estrogen-induced bladder outlet obstruction in a novel mouse model.

Disorders of the prostate and lower urinary tract are common in elderly men. We investigated the role of metallothionein-1 (MT1) in prostate carcinogenesis by generating a prostate-specific, MT1-expressing mouse. Unexpectedly, genomic analyses revealed that a 12.1-kb genomic region harboring several conserved noncoding elements was unintentionally deleted, upstream of the transgene integration site in the mouse, which we named it 12.1ΔMT1. Male 12.1ΔMT1 mice chronically treated with testosterone (T) plus 17ß-estradiol (E2) to induce prostate cancer exhibited no evidence of precancerous or cancerous lesions. Instead, most of them exhibited a bladder outlet obstruction (BOO) phenotype not observed in treated wild-type (WT) mice. Thus, we hypothesized that 12.1ΔMT1 is a novel model for studying the hormonal requirement for BOO induction. Adult male 12.1ΔMT1 and WT mice were treated with T, E2, bisphenol A (BPA), T+E2, or T+BPA for up to 6 months. Histologic and immunohistochemical analysis of the prostate, bladder, and urethra were performed. No significant prostate pathologies were observed in WT or 12.1ΔMT1 mice treated with any of the hormone regimens. As expected, prostatic regression occurred in all E2-treated animals (WT and 12.1ΔMT1). Of great interest, despite a small prostate, 100% of E2-treated 12.1ΔMT1 mice, but only 40% of E2-treated WT mice, developed severe BOO (P<0.01). In contrast, T+E2 treatment was less effective than E2 treatment in inducing severe BOO in 12.1ΔMT1 mice (68%, P<0.05) and was completely ineffective in WT animals. Similarly, T, BPA, and T+BPA treatments did not induce BOO in either WT or 12.1ΔMT1 mice. The BOO pathology includes a thinner detrusor wall, narrowing of bladder neck and urethral lumen, and basal cell hyperplasia in the bladder body and urethra. These findings indicate that 12.1ΔMT1 mice exhibit enhanced susceptibility to E2-induced BOO that is independent of prostate enlargement but that is attenuated by the conjoint treatment with T.

Quantitative Analysis of Glutamate Receptors in Glial Cells from the Cortex of GFAP/EGFP Mice Following Ischemic Injury: Focus on NMDA Receptors.

Cortical glial cells contain both ionotropic and metabotropic glutamate receptors. Despite several efforts, a comprehensive analysis of the entire family of glutamate receptors and their subunits present in glial cells is still missing. Here, we provide an overall picture of the gene expression of ionotropic (AMPA, kainate, NMDA) and the main metabotropic glutamate receptors in cortical glial cells isolated from GFAP/EGFP mice before and after focal cerebral ischemia. Employing single-cell RT-qPCR, we detected the expression of genes encoding subunits of glutamate receptors in GFAP/EGFP-positive (GFAP/EGFP(+)) glial cells in the cortex of young adult mice. Most of the analyzed cells expressed mRNA for glutamate receptor subunits, the expression of which, in most cases, even increased after ischemic injury. Data analyses disclosed several classes of GFAP/EGFP(+) glial cells with respect to glutamate receptors and revealed in what manner their expression correlates with the expression of glial markers prior to and after ischemia. Furthermore, we also examined the protein expression and functional significance of NMDA receptors in glial cells. Immunohistochemical analyses of all seven NMDA receptor subunits provided direct evidence that the GluN3A subunit is present in GFAP/EGFP(+) glial cells and that its expression is increased after ischemia. In situ and in vitro Ca(2+) imaging revealed that Ca(2+) elevations evoked by the application of NMDA were diminished in GFAP/EGFP(+) glial cells following ischemia. Our results provide a comprehensive description of glutamate receptors in cortical GFAP/EGFP(+) glial cells and may serve as a basis for further research on glial cell physiology and pathophysiology.

New N-aryl-N'-(3-(substituted)phenyl)-N'-methylguanidines as leads to potential PET radioligands for imaging the open NMDA receptor.

An expansive set of N-aryl-N'-(3-(substituted)phenyl)-N'-methylguanidines was prepared in a search for new leads to prospective PET ligands for imaging of the open channel of the N-methyl-d-aspartate (NMDA) receptor in vivo. The N-aryl rings and their substituents were varied, whereas the N-methyl group was maintained as a site for potential labeling with the positron-emitter, carbon-11 (t1/2=20.4min). At micromolar concentration, over half of the prepared compounds strongly inhibited the binding of [(3)H]TCP to its binding site in the open NMDA receptor in vitro. Four ligands displayed affinities that are similar or superior to those of the promising SPECT radioligand ([(123)I]CNS1261). The 3'-dimethylamino (19; Ki 36.7nM), 3'-trifluoromethyl (20; Ki 18.3nM) and 3'-methylthio (2; Ki 39.8nM) derivatives of N-1-naphthyl-N'-(phenyl)-N'-methylguanidine were identified as especially attractive leads for PET radioligand development.