Wakefulness-Promoting Effects of Lateral Hypothalamic Area-Deep Brain Stimulation in Traumatic Brain Injury-Induced Comatose Rats: Upregulation of α1-Adrenoceptor Subtypes and Downregulation of Gamma-Aminobutyric Acid ß Receptor Expression Via the Orexins Pathway.

OBJECTIVE: Previous studies have shown that deep brain stimulation (DBS) can improve the level of consciousness of comatose patients with traumatic brain injuries (TBIs). However, the most suitable targets for DBS are unknown, and the mechanisms underlying recovery remain to be determined. The aim of the present study was to assess the effects of lateral hypothalamic area-DBS (LHA-DBS) in comatose rats with TBIs. METHODS: A total of 55 Sprague-Dawley rats were randomly assigned to 5 groups: the control group, TBI group, stimulated (TBI+LHA-DBS) group, antagonist (TBI+SB334867+LHA-DBS) group, and antagonist control (TBI+saline+LHA-DBS) group. The rats in the control group had undergone a sham operation and anesthesia, without coma induction. Coma was induced using a free-fall drop method. The rats in the stimulated group received bilateral LHA stimulation (frequency, 200 Hz; voltage, 2-4 V; pulse width, 0.1 ms) for 1 hour, with 5-minute intervals between subsequent stimulations, which were applied alternately to the left and right sides of the lateral hypothalamus. The comatose rats in the antagonist group received an intracerebroventricular injection with an orexins receptor type 1 (OX1R) antagonist (SB334867) and then received LHA-DBS. A I-VI consciousness scale and electroencephalography were used to assess the level of consciousness in each group of rats after LHA-DBS. Western blotting and immunofluorescence were used to detect OX1R expression in the LHA and α1-adrenoceptor (α1-AR) subtype and gamma-aminobutyric acid ß receptor (GABABR) expression in the prefrontal cortex. RESULTS: In the TBI, stimulated, antagonist, and antagonist control groups, 5, 10, 6, and 9 rats were awakened. The electroencephalographic readings indicated that the proportion of δ waves was lower in the stimulated group than in the TBI and antagonist groups (P < 0.05). Western blotting and immunofluorescence analysis showed that OX1R expression was greater in the stimulated group than in the TBI group (P < 0.05). The expression of α1-AR was also greater in the stimulated group than in the TBI and antagonist groups (P < 0.05). In contrast, the GABABR levels in the stimulated group were lower than those in the TBI and antagonist groups (P < 0.05). A statistically significant difference was found between the antagonist and antagonist control groups. CONCLUSIONS: Taken together, these results suggest that LHA-DBS promotes the recovery of consciousness in comatose rats with TBIs. Upregulation of α1-AR expression and downregulation of GABABR expression in the prefrontal cortex via the orexins and OX1R pathways might be involved in the wakefulness-promoting effects of LHA-DBS.

Microglial activation in the right amygdala-entorhinal-hippocampal complex is associated with preserved spatial learning in AppNL-G-F mice.

BACKGROUND: In Alzheimer`s disease (AD), regional heterogeneity of ß-amyloid burden and microglial activation of individual patients is a well-known phenomenon. Recently, we described a high incidence of inter-individual regional heterogeneity in terms of asymmetry of plaque burden and microglial activation in ß-amyloid mouse models of AD as assessed by positron-emission-tomography (PET). We now investigate the regional associations between amyloid plaque burden, microglial activation, and impaired spatial learning performance in transgenic mice in vivo. METHODS: In 30 AppNL-G-F mice (15 female, 15 male) we acquired cross-sectional 18 kDa translocator protein (TSPO-PET, 18F-GE-180) and ß-amyloid-PET (18F-florbetaben) scans at ten months of age. Control data were obtained from age- and sex-matched C57BI/6 wild-type mice. We assessed spatial learning (i.e. Morris water maze) within two weeks of PET scanning and correlated the principal component of spatial learning performance scores with voxel-wise ß-amyloid and TSPO tracer uptake maps in AppNL-G-F mice, controlled for age and sex. In order to assess the effects of hemispheric asymmetry, we also analyzed correlations of spatial learning performance with tracer uptake in bilateral regions of interest for frontal cortex, entorhinal/piriform cortex, amygdala, and hippocampus, using a regression model. We tested the correlation between regional asymmetry of PET biomarkers with individual spatial learning performance. RESULTS: Voxel-wise analyses in AppNL-G-F mice revealed that higher TSPO-PET signal in the amygdala, entorhinal and piriform cortices, the hippocampus and the hypothalamus correlated with spatial learning performance. Region-based analysis showed significant correlations between TSPO expression in the right entorhinal/piriform cortex and the right amygdala and spatial learning performance, whereas there were no such correlations in the left hemisphere. Right lateralized TSPO expression in the amygdala predicted better performance in the Morris water maze (ß = -0.470, p = 0.013), irrespective of the global microglial activation and amyloid level. Region-based results for amyloid-PET showed no significant associations with spatial learning. CONCLUSION: Elevated microglial activation in the right amygdala-entorhinal-hippocampal complex of AppNL-G-F mice is associated with better spatial learning. Our findings support a protective role of microglia on cognitive function when they highly express TSPO in specific brain regions involved in spatial memory.

Astrocytic TSPO Upregulation Appears Before Microglial TSPO in Alzheimer's Disease.

BACKGROUND: In vivo PET/SPECT imaging of neuroinflammation is primarily based on the estimation of the 18 kDa-translocator-protein (TSPO). However, TSPO is expressed by different cell types which complicates the interpretation. OBJECTIVE: The present study evaluates the cellular origin of TSPO alterations in Alzheimer's disease (AD). METHODS: The TSPO cell origin was evaluated by combining radioactive imaging approaches using the TSPO radiotracer [125I]CLINDE and fluorescence-activated cell sorting, in a rat model of AD (TgF344-AD) and in AD subjects. RESULTS: In the hippocampus of TgF344-AD rats, TSPO overexpression not only concerns glial cells but the increase is visible at 12 and 24 months in astrocytes and only at 24 months in microglia. In the temporal cortex of AD subjects, TSPO upregulation involved only glial cells. However, the mechanism of this upregulation appears different with an increase in the number of TSPO binding sites per cell without cell proliferation in the rat, and a microglial cell population expansion with a constant number of binding sites per cell in human AD. CONCLUSION: These data indicate an earlier astrocyte intervention than microglia and that TSPO in AD probably is an exclusive marker of glial activity without interference from other TSPO-expressing cells. This observation indicates that the interpretation of TSPO imaging depends on the stage of the pathology, and highlights the particular role of astrocytes.

Longitudinal TSPO expression in tau transgenic P301S mice predicts increased tau accumulation and deteriorated spatial learning.

BACKGROUND: P301S tau transgenic mice show age-dependent accumulation of neurofibrillary tangles in the brainstem, hippocampus, and neocortex, leading to neuronal loss and cognitive deterioration. However, there is hitherto only sparse documentation of the role of neuroinflammation in tau mouse models. Thus, we analyzed longitudinal microglial activation by small animal 18 kDa translocator protein positron-emission-tomography (TSPO µPET) imaging in vivo, in conjunction with terminal assessment of tau pathology, spatial learning, and cerebral glucose metabolism. METHODS: Transgenic P301S (n = 33) and wild-type (n = 18) female mice were imaged by 18F-GE-180 TSPO µPET at the ages of 1.9, 3.9, and 6.4 months. We conducted behavioral testing in the Morris water maze, 18F-fluordesoxyglucose (18F-FDG) µPET, and AT8 tau immunohistochemistry at 6.3-6.7 months. Terminal microglial immunohistochemistry served for validation of TSPO µPET results in vivo, applying target regions in the brainstem, cortex, cerebellum, and hippocampus. We compared the results with our historical data in amyloid-ß mouse models. RESULTS: TSPO expression in all target regions of P301S mice increased exponentially from 1.9 to 6.4 months, leading to significant differences in the contrasts with wild-type mice at 6.4 months (+ 11-23%, all p < 0.001), but the apparent microgliosis proceeded more slowly than in our experience in amyloid-ß mouse models. Spatial learning and glucose metabolism of AT8-positive P301S mice were significantly impaired at 6.3-6.5 months compared to the wild-type group. Longitudinal increases in TSPO expression predicted greater tau accumulation and lesser spatial learning performance at 6.3-6.7 months. CONCLUSIONS: Monitoring of TSPO expression as a surrogate of microglial activation in P301S tau transgenic mice by µPET indicates a delayed time course when compared to amyloid-ß mouse models. Detrimental associations of microglial activation with outcome parameters are opposite to earlier data in amyloid-ß mouse models. The contribution of microglial response to pathology accompanying amyloid-ß and tau over-expression merits further investigation.

Translocator protein localises to CD11b+ macrophages in atherosclerosis.

BACKGROUND AND AIMS: Atherosclerosis is characterized by lipid deposition, monocyte infiltration and foam cell formation in the artery wall. Translocator protein (TSPO) is abundantly expressed in lipid rich tissues. Recently, TSPO has been identified as a potential diagnostic tool in cardiovascular disease. The purpose of this study was to determine if the TSPO ligand, 18F-PBR111, can identify early atherosclerotic lesions and if TSPO expression can be used to identify distinct macrophage populations during lesion progression. METHODS: ApoE-/- mice were maintained on a high-fat diet for 3 or 12 weeks. C57BL/6J mice maintained on chow diet served as controls. Mice were administered 18F-PBR111 intravenously and PET/CT imaged. After euthanasia, aortas were isolated, fixed and optically cleared. Cleared aortas were immunostained with DAPI, and fluorescently labelled with antibodies to TSPO, the tissue resident macrophage marker F4/80 and the monocyte-derived macrophage marker CD11b. TSPO expression and the macrophage markers were visualised in fatty streaks and established plaques by light sheet microscopy. RESULTS: While tissue resident F4/80 + macrophages were evident in the arteries of animals without atherosclerosis, no CD11b + macrophages were observed in these animals. In contrast, established plaques had high CD11b and low F4/80 expression. A ∼3-fold increase in the uptake of 18F-PBR111 was observed in the aortas of atherosclerotic mice relative to controls. CONCLUSIONS: Imaging of TSPO expression is a new approach for studying atherosclerotic lesion progression and inflammatory cell infiltration. The TSPO ligand, 18F-PBR111, is a potential clinical diagnostic tool for the detection and quantification of atherosclerotic lesion progression in humans.

Cell injury and receptor expression in the epileptic human amygdala.

Neuropathological findings in the amygdala obtained from patients with mesial temporal lobe epilepsy (MTLE) indicate varying degrees of histopathological alterations, such as neuronal loss and gliosis. The mechanisms underlying cellular damage in the amygdala of patients with MTLE have not been fully elucidated. In the present study, we assess cellular damage, determine the receptor expression of major inhibitory and excitatory neurotransmitters, and evaluate the correlation between the expression of various receptors and cell damage in the basolateral complex and the centromedial areas in the amygdala specimens resected during brain surgery on 30 patients with medically intractable MTLE. Our data reveal an increased rate of cell damage and apoptosis as well as decreased expression levels of several GABAergic receptor subunits (GABAARα1, GABAARß3, and GABABR1) and GAD65 in the amygdalae obtained during epilepsy surgery compared to autopsy specimens. Analyses of the expression of glutamate excitatory receptor subunits (NR1, NR2B, mGluR1α, GluR1, and GluR2) reveal no significant differences between the epileptic amygdalae and autopsy control tissues. Furthermore, the increased occurrence of apoptotic cells in the amygdala is negatively correlated with the reduced expression of the studied GABAergic receptor subunits and GAD65 but is not correlated with the expression of excitatory receptors. The present data point to the importance of GABAergic neurotransmission in seizure-induced cell injury in the amygdala of patients with MTLE and suggest several GABA receptor subunits as potential druggable target structures to control epilepsy and its comorbid disorders, such as anxiety.

TSPO expression and brain structure in the psychosis spectrum.

Psychosis is associated with abnormal structural changes in the brain including decreased regional brain volumes and abnormal brain morphology. However, the underlying causes of these structural abnormalities are less understood. The immune system, including microglial activation, has been implicated in the pathophysiology of psychosis. Although previous studies have suggested a connection between peripheral proinflammatory cytokines and structural brain abnormalities in schizophrenia, no in-vivo studies have investigated whether microglial activation is also linked to brain structure alterations previously observed in schizophrenia and its putative prodrome. In this study, we investigated the link between mitochondrial 18 kDa translocator protein (TSPO) and structural brain characteristics (i.e. regional brain volume, cortical thickness, and hippocampal shape) in key brain regions such as dorsolateral prefrontal cortex and hippocampus of a large group of participants (N = 90) including individuals at clinical high risk (CHR) for psychosis, first-episode psychosis (mostly antipsychotic-naïve) patients, and healthy volunteers. The participants underwent structural brain MRI scan and [18F]FEPPA positron emission tomography (PET) targeting TSPO. A significant [18F]FEPPA binding-by-group interaction was observed in morphological measures across the left hippocampus. In first-episode psychosis, we observed associations between [18F]FEPPA VT (total volume of distribution) and outward and inward morphological alterations, respectively, in the dorsal and ventro-medial portions of the left hippocampus. These associations were not significant in CHR or healthy volunteers. There was no association between [18F]FEPPA VT and other structural brain characteristics. Our findings suggest a link between TSPO expression and alterations in hippocampal morphology in first-episode psychosis.

Positron emission tomography of cerebral angiogenesis and TSPO expression in a mouse model of chronic hypoxia.

The present study aimed to examine whether positron emission tomography (PET) could evaluate cerebral angiogenesis. Mice were housed in a hypoxic chamber with 8-9% oxygen for 4, 7, and 14 days, and the angiogenic responses were evaluated with a radiotracer, 64Cu-cyclam-RAFT-c(-RGDfK-)4, which targeted αVß3 integrin and was imaged with PET. The PET imaging results showed little uptake during all of the hypoxic periods. Immunofluorescence staining of the ß3 integrin, CD61, revealed weak expression, while the microvessel density assessed by CD31 staining increased with the hypoxic duration. These observations suggest that the increased vascular density originated from other types of vascular remodeling, unlike angiogenic sprouting. We then searched for any signs of vascular remodeling that could be detected using PET. PET imaging of 11C-PK11195, a marker of the 18-kDa translocator protein (TSPO), revealed a transient increase at day 4 of hypoxia. Because the immunofluorescence of glial markers showed unchanged staining over the early phase of hypoxia, the observed upregulation of TSPO expression probably originated from non-glial cells (e.g. vascular cells). In conclusion, a transient increase in TSPO probe uptake was detected with PET at only the early phase of hypoxia, which indicates an early sign of vascular remodeling induced by hypoxia.

2-Cl-MGV-1 Ameliorates Apoptosis in the Thalamus and Hippocampus and Cognitive Deficits After Cortical Infarct in Rats.

BACKGROUND AND PURPOSE: Focal cortical infarction causes neuronal apoptosis in the ipsilateral nonischemic thalamus and hippocampus, which is potentially associated with poststroke cognitive deficits. TSPO (translocator protein) is critical in regulating mitochondrial apoptosis pathways. We examined the effects of the novel TSPO ligand 2-(2-chlorophenyl) quinazolin-4-yl dimethylcarbamate (2-Cl-MGV-1) on poststroke cognitive deficits, neuronal mitochondrial apoptosis, and secondary damage in the ipsilateral thalamus and hippocampus after cortical infarction. METHODS: One hundred fourteen hypertensive rats underwent successful distal middle cerebral artery occlusion (n=76) or sham procedures (n=38). 2-Cl-MGV-1 or dimethyl sulfoxide as vehicle was administrated 2 hours after distal middle cerebral artery occlusion and then for 6 or 13 days (n=19 per group). Spatial learning and memory were tested using the Morris water maze. Secondary degeneration and mitochondrial apoptosis in the thalamus and hippocampus were assessed using Nissl staining, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling, JC-1 staining, and immunoblotting 7 and 14 days after surgery. RESULTS: Infarct volumes did not significantly differ between the vehicle and 2-Cl-MGV-1 groups. There were more neurons and fewer glia in the ipsilateral thalamus and hippocampus in the vehicle groups than in the sham-operated group 7 and 14 days post-distal middle cerebral artery occlusion. 2-Cl-MGV-1 significantly ameliorated spatial cognitive impairment and decreased neuronal death and glial activation when compared with vehicle treatment (P<0.05). The collapse of mitochondrial transmembrane potential and cytoplasmic release of apoptosis-inducing factors and cytochrome c was prevented within the thalamus. Caspase cleavage and the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling+ or Nissl atrophic cells were reduced within the thalamus and hippocampus. This was accompanied by upregulation of B-cell lymphoma 2 and downregulation of Bax (P<0.05). CONCLUSIONS: 2-Cl-MGV-1 reduces neuronal apoptosis via mitochondrial-dependent pathways and attenuates secondary damage in the nonischemic thalamus and hippocampus, potentially contributing to ameliorated cognitive deficits after cortical infarction.

Comparison of γ-Aminobutyric Acid, Type A (GABAA), Receptor αßγ and αßδ Expression Using Flow Cytometry and Electrophysiology: EVIDENCE FOR ALTERNATIVE SUBUNIT STOICHIOMETRIES AND ARRANGEMENTS.

The subunit stoichiometry and arrangement of synaptic αßγ GABAA receptors are generally accepted as 2α:2ß:1γ with a ß-α-γ-ß-α counterclockwise configuration, respectively. Whether extrasynaptic αßδ receptors adopt the analogous ß-α-δ-ß-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or ß2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and ß2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of ß2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1ß2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αßγ and αßδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αßγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical ß-α-γ/δ-ß-α receptors, including ß-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and ß-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABAA receptor subunit under- or overexpression in disease states such as genetic epilepsies.

Analysis of persistent changes to γ-aminobutyric acid receptor gene expression in Plutella xylostella subjected to sublethal amounts of spinosad.

A multi-generational approach was used to investigate the persistent effects of a sub-lethal dose of spinosad in Plutella xylostella. The susceptibility of various sub-populations of P. xylostella to spinosad and the effects of the insecticide on the gene expression of γ-aminobutyric acid receptor (GABAR) were determined. The results of a leaf dip bioassay showed that the sensitivity of P. xylostella to spinosad decreased across generations. The sub-strains had been previously selected based on a determined LC25 of spinosad. Considering that GABA-gated chloride channels are the primary targets of spinosad, the cDNA of P. xylostella was used to clone GABARα by using reverse transcription-polymerase chain reaction (RT-PCR). The mature peptide cDNA was 1477-bp long and contained a 1449-bp open reading frame encoding a protein of 483 amino acids. The resulting amino acid sequence was used to generate a neighbor-joining dendrogram, and homology search was conducted using NCBI BLAST. The protein had high similarity with the known GABAR sequence from P. xylostella. Subsequent semi-quantitative RT-PCR and real-time PCR analyses indicated that the GABAR transcript levels in the spinosad-resistant strain (RR, 145.82-fold) and in Sub1 strain (selected with LC25 spinosad for one generation) were the highest, followed by those in the spinosad-susceptible strain, the Sub10 strain (selected for ten generations), and the Sub5 strain (selected for five generations). This multi-generational study found significant correlations between spinosad susceptibility and GABAR gene expression, providing insights into the long-term effects of sub-lethal insecticide exposure and its potential to lead to the development of insecticide-resistant insect populations.

Augmented expression of TSPO after intracerebral hemorrhage: a role in inflammation?

BACKGROUND: Intracerebral hemorrhage (ICH) is a potentially fatal stroke subtype accounting for 10-15 % of all strokes. Despite neurosurgical intervention and supportive care, the 30-day mortality rate remains 30-50 % with ICH survivors frequently displaying neurological impairment and requiring long-term assisted care. Although accumulating evidence demonstrates the role of neuroinflammation in secondary brain injury and delayed fatality after ICH, the molecular regulators of neuroinflammation remain poorly defined after ICH. METHODS: In the present study, ICH was induced in CD1 male mice by collagenase injection method and given the emerging role of TSPO (18-kDa translocator protein) in neuroinflammation, immunofluorescence staining of brain sections was performed to characterize the temporal expression pattern and cellular and subcellular localization of TSPO after ICH. Further, both genetic and pharmacological studies were employed to assess the functional role of TSPO in neuroinflammation. RESULTS: The expression of TSPO was found to be increased in the peri-hematomal brain region 1 to 7 days post-injury, peaking on day 3 to day 5 in comparison to sham. Further, the TSPO expression was mostly observed in microglia/macrophages, the inflammatory cells of the central nervous system, suggesting an unexplored role of TSPO in neuroinflammatory responses after ICH. Further, the subcellular localization studies revealed prominent perinuclear expression of TSPO after ICH. Moreover, both genetic and pharmacological studies revealed a regulatory role of TSPO in the release of pro-inflammatory cytokines in a macrophage cell line, RAW 264.7. CONCLUSIONS: Altogether, the data suggest that TSPO induction after ICH could be an intrinsic mechanism to prevent an exacerbated inflammatory response and raise the possibility of targeting TSPO for the attenuation of secondary brain injury after ICH.

Translocator Protein-Mediated Stabilization of Mitochondrial Architecture during Inflammation Stress in Colonic Cells.

UNLABELLED: Chronic inflammation of the gastrointestinal tract increasing the risk of cancer has been described to be linked to the high expression of the mitochondrial translocator protein (18 kDa; TSPO). Accordingly, TSPO drug ligands have been shown to regulate cytokine production and to improve tissue reconstruction. We used HT-29 human colon carcinoma cells to evaluate the role of TSPO and its drug ligands in tumor necrosis factor (TNF)-induced inflammation. TNF-induced interleukin (IL)-8 expression, coupled to reactive oxygen species (ROS) production, was followed by TSPO overexpression. TNF also destabilized mitochondrial ultrastructure, inducing cell death by apoptosis. Treatment with the TSPO drug ligand PK 11195 maintained the mitochondrial ultrastructure, reducing IL-8 and ROS production and cell death. TSPO silencing and overexpression studies demonstrated that the presence of TSPO is essential to control IL-8 and ROS production, so as to maintain mitochondrial ultrastructure and to prevent cell death. Taken together, our data indicate that inflammation results in the disruption of mitochondrial complexes containing TSPO, leading to cell death and epithelia disruption. SIGNIFICANCE: This work implicates TSPO in the maintenance of mitochondrial membrane integrity and in the control of mitochondrial ROS production, ultimately favoring tissue regeneration.

Sildenafil reduces neuroinflammation and restores spatial learning in rats with hepatic encephalopathy: underlying mechanisms.

BACKGROUND: There are no specific treatments for the neurological alterations of cirrhotic patients with minimal hepatic encephalopathy (MHE). Rats with MHE due to portacaval shunt (PCS) show impaired spatial learning. The underlying mechanisms remain unknown. The aims of this work were to assess: (a) whether PCS rats show neuroinflammation in hippocampus, (b) whether treatment with sildenafil reduces neuroinflammation and restores spatial learning in PCS rats, and (c) analyze the underlying mechanisms. METHODS: Neuroinflammation was assessed by determining inflammatory markers by Western blot. Phosphorylation of MAP-kinase p38 was assessed by immunohistochemistry. Membrane expression of GABA and glutamate receptors was analyzed using BS3 cross-linker. Spatial learning was analyzed using the radial and Morris water mazes. To assess if sildenafil reverses the alterations, rats were treated with sildenafil in the drinking water. RESULTS: PCS rats show increased IL-1ß and TNF-α levels and phosphorylation (activity) of p38 in hippocampus. Membrane expression of subunits α1 of GABAA receptor and GluR2 of AMPA receptor are increased in PCS rats, while subunits GluR1 of AMPA receptors and NR1 and NR2a of NMDA receptors are reduced. PCS rats show reduced spatial learning in the radial and Morris water mazes. Sildenafil treatment normalizes IL-1ß and TNF-α levels, p38 phosphorylation, and membrane expression of GABAA, AMPA, and NMDA receptors and restores spatial learning. CONCLUSIONS: Increased IL-1ß alters GABAergic and glutamatergic neurotransmission in hippocampus and impairs spatial learning in rats with MHE. Sildenafil reduces neuroinflammation and restores learning. Phosphodiesterase-5 inhibitors may be useful to improve cognitive function in patients with MHE.

In vivo electroretinographic studies of the role of GABAC receptors in retinal signal processing.

All three classes of receptors for the inhibitory neurotransmitter GABA (GABAR) are expressed in the retina. This study investigated roles of GABAR, especially GABACR (GABA(A)-ρ), in retinal signaling in vivo by studying effects on the mouse electroretinogram (ERG) of genetic deletion of GABACR versus pharmacological blockade using receptor antagonists. Brief full-field flash ERGs were recorded from anesthetized GABACR(-/-) mice, and WT C57BL/6 (B6) mice, before and after intravitreal injection of GABACR antagonists, TPMPA, 3-APMPA, or the more recently developed 2-AEMP; GABAAR antagonist, SR95531; GABABR antagonist, CGP, and agonist, baclofen. Intravitreal injections of TPMPA and SR95531 were also made in Brown Norway rats. The effect of 2-AEMP on GABA-induced current was tested directly in isolated rat rod bipolar cells, and 2-AEMP was found to preferentially block GABACR in those cells. Maximum amplitudes of dark (DA) and light-adapted (LA) ERG b-waves were reduced in GABACR(-/-) mice, compared to B6 mice, by 30-60%; a-waves were unaltered and oscillatory potential amplitudes were increased. In B6 mice, after injection of TPMPA (also in rats), 3-APMPA or 2-AEMP, ERGs became similar to ERGs of GABACR(-/-) mice. Blockade of GABAARs and GABABRs, or agonism of GABABRs did not alter B6 DA b-wave amplitude. The negative scotopic threshold response (nSTR) was slightly less sensitive in GABACR(-/-) than in B6 mice, and unaltered by 2-AEMP. However, amplitudes of nSTR and photopic negative response (PhNR), both of which originate from inner retina, were enhanced by TPMPA and 3-APMPA, each of which has GABAB agonist properties, and further increased by baclofen. The finding that genetic deletion of GABACR, the GABACR antagonist 2-AEMP, and other antagonists all reduced ERG b-wave amplitude, supports a role for GABACR in determining the maximum response amplitude of bipolar cells contributing to the b-wave. GABACR antagonists differed in their effects on nSTR and PhNR; antagonists with GABAB agonist properties enhanced light-driven responses whereas 2-AEMP did not.

Direct conversion of mouse fibroblasts to GABAergic neurons with combined medium without the introduction of transcription factors or miRNAs.

Degeneration or loss of GABAergic neurons frequently may lead to many neuropsychiatric disorders such as epilepsy and autism spectrum disorders. So far no clinically effective therapies can slow and halt the progression of these diseases. Cell-replacement therapy is a promising strategy for treatment of these neuropsychiatric diseases. Although increasing evidence showed that mammalian somatic cells can be directly converted into functional neurons using specific transcription factors or miRNAs via virus delivery, the application of these induced neurons is potentially problematic, due to integration of vectors into the host genome, which results in the disruption or dysfunction of nearby genes. Here, we show that mouse fibroblasts could be efficiently reprogrammed into GABAergic neurons in a combined medium composed of conditioned medium from neurotrophin-3 modified Olfactory Ensheathing Cells (NT3-OECs) plus SB431542, GDNF and RA. Following 3 weeks of induction, these cells derived from fibroblasts acquired the morphological and phenotypical GABAerigic neuronal properties, as demonstrated by the expression of neuronal markers including Tuj1, NeuN, Neurofilament-L, GABA, GABA receptors and GABA transporter 1. More importantly, these converted cells acquired neuronal functional properties such as synapse formation and increasing intracellular free calcium influx when treated with BayK, a specific activator of L-type calcium channel. Therefore, our findings demonstrate for the first time that fibroblasts can be directly converted into GABAergic neurons without ectopic expression of specific transcription factors or miRNA. This study may provide a promising cell source for the application of cell replacement therapy in neuropsychiatric disorders.

The Effect of Cigarette Smoke on the Translocator Protein (TSPO) in Cultured Lung Cancer Cells.

Lung cancer is prevalent in cigarette smokers. The mitochondrial membrane translocator protein (TSPO), is thought to protect cells from free radical damage. We examined the effect of cigarette smoke (CS) (containing free radicals) alone and in the presence of saliva (containing redox active free iron), on survival of H1299 lung cancer cells and on their mitochondrial characteristics, and whether TSPO binding was influenced by CS and by saliva. We exposed H1299 cells to CS in the presence/absence of saliva and also characterized TSPO binding in the cells using [3H]PK 11195 as a radioligand. CS induced a significant drop in mitochondrial potential (ΔΨm), while addition of saliva did not lead to further loss of ΔΨm (42.5% vs. 39.85%). Scatchard analysis of the saturation curve of [3H]PK 11195 binding (0.2-6 nM final concentration) yielded a straight-line plot (R = 0.9). Average Bmax value was 3274 ± 787 fmol/mg of protein, and average Kd value was 9.2 ± 1.3 nM. Benzodiazepine diazepam partially prevented decrease in cell survival following exposure to CS and redox active iron containing media (saliva) while benzodiazepine clonazepam did not, indicating that this effect is TSPO-specific. Exposure of cells to CS resulted in alternation of biomolecules expressed by CLs peroxidation, reduction of TSPO binding, and depletion of the mitochondrial potential. This irreversible damage was enhanced in the presence of saliva. All these modulations may result in cellular death increase following CS exposure, enhanced in the presence of saliva.

Facile synthesis of SSR180575 and discovery of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[(18)F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide, a potent pyridazinoindole ligand for PET imaging of TSPO in cancer.

A novel synthesis of the translocator protein (TSPO) ligand 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (SSR180575, 3) was achieved in four steps from commercially available starting materials. Focused structure-activity relationship development about the pyridazinoindole ring at the N3 position led to the discovery of 7-chloro-N,N,5-trimethyl-4-oxo-3(6-fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (14), a novel ligand of comparable affinity. Radiolabeling with fluorine-18 ((18)F) yielded 7-chloro-N,N,5-trimethyl-4-oxo-3(6-[(18)F]fluoropyridin-2-yl)-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide ([(18)F]-14) in high radiochemical yield and specific activity. In vivo studies of [(18)F]-14 revealed this agent as a promising probe for molecular imaging of glioma.

Kinetic modeling without accounting for the vascular component impairs the quantification of [(11)C]PBR28 brain PET data.

The positron emission tomography radioligand [(11)C]PBR28 targets translocator protein (18 kDa) (TSPO) and is a potential marker of neuroinflammation. [(11)C]PBR28 binding is commonly quantified using a two-tissue compartment model and an arterial input function. Previous studies with [(11)C]-(R)-PK11195 demonstrated a slow irreversible binding component to the TSPO proteins localized in the endothelium of brain vessels, such as venous sinuses and arteries. However, the impact of this component on the quantification of [(11)C]PBR28 data has never been investigated. In this work we propose a novel kinetic model for [(11)C]PBR28. This model hypothesizes the existence of an additional irreversible component from the blood to the endothelium. The model was tested on a data set of 19 healthy subjects. A simulation was also performed to quantify the error generated by the standard two-tissue compartmental model when the presence of the irreversible component is not taken into account. Our results show that when the vascular component is included in the model the estimates that include the vascular component (2TCM-1K) are more than three-fold smaller, have a higher time stability and are better correlated to brain mRNA TSPO expression than those that do not include the model (2TCM).