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.

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.

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.

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.

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.

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.

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.

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.

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.

Altered thalamic GABAA-receptor subunit expression in the stargazer mouse model of absence epilepsy.

PURPOSE: Absence seizures, also known as petit mal seizures, arise from disruptions within the cortico-thalamocortical network. Interconnected circuits within the thalamus consisting of inhibitory neurons of the reticular thalamic nucleus (RTN) and excitatory relay neurons of the ventral posterior (VP) complex, generate normal intrathalamic oscillatory activity. The degree of synchrony in this network determines whether normal (spindle) or pathologic (spike wave) oscillations occur; however, the cellular and molecular mechanisms underlying absence seizures are complex and multifactorial and currently are not fully understood. Recent experimental evidence from rodent models suggests that regional alterations in γ-aminobutyric acid (GABA)ergic inhibition may underlie hypersynchronous oscillations featured in absence seizures. The aim of the current study was to investigate whether region-specific differences in GABAA receptor (GABAAR) subunit expression occur in the VP and RTN thalamic regions in the stargazer mouse model of absence epilepsy where the primary deficit is in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression. METHODS: Immunofluorescence confocal microscopy and semiquantitative Western blot analysis were used to investigate region-specific changes in GABAAR subunits in the thalamus of the stargazer mouse model of absence epilepsy to determine whether changes in GABAergic inhibition could contribute to the mechanisms underlying seizures in this model of absence epilepsy. KEY FINDINGS: Immunofluorescence confocal microscopy revealed that GABAAR α1 and ß2 subunits are predominantly expressed in the VP, whereas α3 and ß3 subunits are localized primarily in the RTN. Semiquantitative Western blot analysis of VP and RTN samples from epileptic stargazers and their nonepileptic littermates showed that GABAAR α1 and ß2 subunit expression levels in the VP were significantly increased (α1: 33%, ß2: 96%) in epileptic stargazers, whereas α3 and ß3 subunits in the RTN were unchanged in the epileptic mice compared to nonepileptic control littermates. SIGNIFICANCE: These findings suggest that region-specific differences in GABAAR subunits in the thalamus of epileptic mice, specifically up-regulation of GABAARs in the thalamic relay neurons of the VP, may contribute to generation of hypersynchronous thalamocortical activity in absence seizures. Understanding region-specific differences in GABAAR subunit expression could help elucidate some of the cellular and molecular mechanisms underlying absence seizures and thereby identify targets by which drugs can modulate the frequency and severity of epileptic seizures. Ultimately, this information could be crucial for the development of more specific and effective therapeutic drugs for treatment of this form of epilepsy.

Co-expression of γ2 subunits hinders processing of N-linked glycans attached to the N104 glycosylation sites of GABAA receptor ß2 subunits.

GABAA receptors, the major mediators of fast inhibitory neuronal transmission, are heteropentameric glycoproteins assembled from a panel of subunits, usually including α and ß subunits with or without a γ2 subunit. The α1ß2γ2 receptor is the most abundant GABAA receptor in brain. Co-expression of γ2 with α1 and ß2 subunits causes conformational changes, increases GABAA receptor channel conductance, and prolongs channel open times. We reported previously that glycosylation of the three ß2 subunit glycosylation sites, N32, N104 and N173, was important for α1ß2 receptor channel gating. Here, we examined the hypothesis that steric effects or conformational changes caused by γ2 subunit co-expression alter the glycosylation of partnering ß2 subunits. We found that co-expression of γ2 subunits hindered processing of ß2 subunit N104 N-glycans in HEK293T cells. This γ2 subunit-dependent effect was strong enough that a decrease of γ2 subunit expression in heterozygous GABRG2 knockout (γ2(+/-)) mice led to appreciable changes in the endoglycosidase H digestion pattern of neuronal ß2 subunits. Interestingly, as measured by flow cytometry, γ2 subunit surface levels were decreased by mutating each of the ß2 subunit glycosylation sites. The ß2 subunit mutation N104Q also decreased GABA potency to evoke macroscopic currents and reduced conductance, mean open time and open probability of single channel currents. Collectively, our data suggested that γ2 subunits interacted with ß2 subunit N-glycans and/or subdomains containing the glycosylation sites, and that γ2 subunit co-expression-dependent alterations in the processing of the ß2 subunit N104 N-glycans were involved in altering the function of surface GABAA receptors.

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.

Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia.

OBJECTIVE: The most common neurological symptom of tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) is early life refractory epilepsy. As previous studies have shown enhanced excitatory glutamatergic neurotransmission in TSC and FCD brains, we hypothesized that neurons associated with these lesions may also express altered γ-aminobutyric acid (GABA)(A) receptor (GABA(A)R)-mediated inhibition. METHODS: Expression of the GABA(A)R subunits α1 and α4, and the Na(+)-K(+)-2Cl(-) (NKCC1) and the K(+)-Cl(-) (KCC2) transporters, in human TSC and FCD type II specimens were analyzed by Western blot and double label immunocytochemistry. GABA(A) R responses in dysplastic neurons from a single case of TSC were measured by perforated patch recording and compared to normal-appearing cortical neurons from a non-TSC epilepsy case. RESULTS: TSC and FCD type IIb lesions demonstrated decreased expression of GABA(A)R α1, and increased NKCC1 and decreased KCC2 levels. In contrast, FCD type IIa lesions showed decreased α4, and increased expression of both NKCC1 and KCC2 transporters. Patch clamp recordings from dysplastic neurons in acute slices from TSC tubers demonstrated excitatory GABA(A)R responses that were significantly attenuated by the NKCC1 inhibitor bumetanide, in contrast to hyperpolarizing GABA(A)R-mediated currents in normal neurons from non-TSC cortical slices. INTERPRETATION: Expression and function of GABA(A)Rs in TSC and FCD type IIb suggest the relative benzodiazepine insensitivity and more excitatory action of GABA compared to FCD type IIa. These factors may contribute to resistance of seizure activity to anticonvulsants that increase GABAergic function, and may justify add-on trials of the NKCC1 inhibitor bumetanide for the treatment of TSC and FCD type IIb-related epilepsy.

Alcohol administration blocks stress-induced impairments in memory and anxiety, and alters hippocampal neurotransmitter receptor expression in male rats.

Chronic exposure to stress has many deleterious effects on behavior, which can often lead to self-medication with anxiolytics, antidepressants, or alcohol. We determined the effects of alcohol administration following a stressor on established behavioral, physiological, and neural responses to stress. Male Sprague-Dawley rats received: No alcohol/No stress (CON), Alcohol alone (ALC), Stress alone (STR), or Stress plus Alcohol (STR+ALC). For seven consecutive days, two cohorts received an oral dose of 2.0 g/kg of either 20% ethanol or saline. In Cohort 1, behavioral testing began after the final treatment (day-8). Memory was tested using the object recognition (OR) and Y-maze, anxiety on the plus maze, and depression on the forced swim task. Memory on OR and Y-maze tasks was impaired in the ALC and STR groups. This deficit was reversed in the STR+ALC group, which performed not differently from the CON group. Stress alone was associated with increased anxiety, which was alleviated with alcohol treatment. No treatment effects were found in the forced swim task. In Cohort 2, hippocampal GABAα4 was upregulated in the STR+ALC group and GluN2B was upregulated in the ALC and STR+ALC groups. The STR+ALC group in Cohort 1 showed enhanced corticosterone levels after forced swim. The STR+ALC group in Cohort 2 showed increased corticosterone levels on day-1 of treatment and a habituation by day-7. In conclusion, this study found a reversal of stress-induced deficits in cognition and anxiety when alcohol was given post-stress, and changes in neurotransmitter receptor expression may contribute to these behavioral effects.

Translocator protein (Tspo) gene promoter-driven green fluorescent protein synthesis in transgenic mice: an in vivo model to study Tspo transcription.

Translocator protein (TSPO), previously known as the peripheral-type benzodiazepine receptor, is a ubiquitous drug- and cholesterol-binding protein primarily found in the outer mitochondrial membrane as part of a mitochondrial cholesterol transport complex. TSPO is present at higher levels in steroid-synthesizing and rapidly proliferating tissues and its biological role has been mainly linked to mitochondrial function, steroidogenesis and cell proliferation/apoptosis. Aberrant TSPO levels have been linked to multiple diseases, including cancer, endocrine disorders, brain injury, neurodegeneration, ischemia-reperfusion injury and inflammatory diseases. Investigation of the functions of this protein in vitro and in vivo have been mainly carried out using high-affinity drug ligands, such as isoquinoline carboxamides and benzodiazepines and more recently, gene silencing methods. To establish a model to study the regulation of Tspo transcription in vivo, we generated a transgenic mouse model expressing green fluorescent protein (GFP) from Aequorea coerulescens under control of the Tspo promoter region (Tspo-AcGFP). The expression profiles of Tspo-AcGFP, endogenous TSPO and Tspo mRNA were found to be well-correlated. Tspo-AcGFP synthesis in the transgenic mice was seen in almost every tissue examined and as with TSPO in wild-type mice, Tspo-AcGFP was highly expressed in steroidogenic cells of the endocrine and reproductive systems, epithelial cells of the digestive system, skeletal muscle and other organs. In summary, this transgenic Tspo-AcGFP mouse model recapitulates endogenous Tspo expression patterns and could be a useful, tractable tool for monitoring the transcriptional regulation and function of Tspo in live animal experiments.

[Apoptosis modulation by PMA occurs independently of TsPO expression in squamous cell carcinoma cells].

Phorbol esters are known to modulate protein kinase C activity - enzyme involved in cell proliferation, differentiation and apoptosis regulation in skin cells. Besides it phorbol-12-myristate 13-acetate was shown possible to modulate promoter activity of TsPO - protein that is involved in steroidogenesis and cell proliferation regulation. Caspase-3 and TsPO expression was measured in squamous cell carcinoma cells after incubation with phorbol-12-myristate 13-acetate and ultraviolet radiation. Following alterations of TsPO and caspase-3 levels are explained by different mechanisms of regulation.

ρ1 GABAC receptors are expressed in fibrous and cartilaginous layers of chick sclera and located on sclera fibroblasts and chondrocytes.

rho(1) GABA(C) receptor antagonists inhibit myopia in chick but the site of this effect is not known. The sclera ultimately determines the shape and size of the globe and thus an untested possibility is that GABA agents have a scleral mechanism. Whether rho(1) GABA(C) receptors are expressed and located in chick sclera is unknown. Real-time PCR, western blot and immunohistochemistry were used to determine whether rho1 GABA(C) receptors are expressed and located in chick fibrous and cartilaginous sclera. Both layers of the chick sclera were positive for rho1 GABA(C) receptor mRNA (PCR) and protein (western blot) expression and labeling was observed in both fibroblasts and chondrocytes of the fibrous and cartilaginous layers (immunohistochemistry). These investigations clearly show that chick sclera possesses rho(1) GABA(C) receptors. The sclera is thus a potential previously unrecognized site for activity of rho(1) GABA(C) agents.

Serotonin transporter (SERT) and translocator protein (TSPO) expression in the obese ob/ob mouse.

BACKGROUND: An ever growing body of evidences is emerging concerning metabolism hormones, neurotransmitters or stress-related biomarkers as effective modulators of eating behavior and body weight in mammals. The present study sought at examining the density and affinity of two proteins related to neurotransmission and cell metabolism, the serotonin transporter SERT and the cholesterol import-benzodiazepine site TSPO (translocator protein), in a rodent leptin-lacking mutant, the obese ob/ob mouse. Binding studies were thus carried out in brain or peripheral tissues, blood platelets (SERT) and kidneys (TSPO), of ob/ob and WT mice supplied with a standard diet, using the selective radiochemical ligands [3H]-paroxetine and [3H]-PK11195. RESULTS: We observed comparable SERT number or affinity in brain and platelets of ob/ob and WT mice, whilst a significantly higher [3H]-PK11195 density was reported in the brain of ob/ob animals. TSPO binding parameters were similar in the kidneys of all tested mice. By [3H]-PK11195 autoradiography of coronal hypothalamic-hippocampal sections, an increased TSPO signal was detected in the dentate gyrus (hippocampus) and choroids plexus of ob/ob mice, without appreciable changes in the cortex or hypothalamic-thalamic regions. CONCLUSIONS: These findings show that TSPO expression is up-regulated in cerebral regions of ob/ob leptin-deficient mice, suggesting a role of the translocator protein in leptin-dependent CNS trophism and metabolism. Unchanged SERT in mutant mice is discussed herein in the context of previous literature as the forerunner to a deeper biochemical investigation.