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Background: Translocator protein (TSPO) is a neuroinflammation hallmark. Different TSPO affinity compounds have been produced and over time, the techniques of radiolabeling have been refined. The aim of this systematic review is to summarize the development of new radiotracers for dementia and neuroinflammation imaging., Methods: An online search of the literature was conducted in the PubMed, Scopus, Medline, Cochrane Library, and Web of Science databases, selecting published studies from January 2004 to December 2022. The accepted studies considered the synthesis of TSPO tracers for nuclear medicine imaging in dementia and neuroinflammation., Results: A total of 50 articles was identified. Twelve papers were selected from the included studies' bibliographies and 34 were excluded. Thus, 28 articles were ultimately selected for quality assessment., Conclusion: Huge efforts in developing specific and stable tracers for PET/SPECT imaging have been made. The long half-life of 18 F makes this isotope a preferable choice to 11 C. An emerging limitation to this however is that neuroinflammation involves all of the brain which inhibits the possibility of detecting a slight inflammation status change in patients. A partial solution to this is using the cerebellum as a reference region and developing higher TSPO affinity tracers. Moreover, it is necessary to consider the presence of distomers and racemic compounds interfering with pharmacological tracers' effects and increasing the noise ratio in images.

Objectives/hypothesis: Tinnitus can develop due to, or be aggravated by, stress in a rat model. To investigate stress as a possible causal factor in the development of tinnitus, we designed an animal study that included tinnitus behavior and excitatory/inhibitory neurotransmitter expression after noise exposure as well as restraint stress., Study Design: An experimental animal study., Methods: Wistar rats were grouped according to single or double exposure to noise and restraint stress. The noise exposure (NE) group was subjected to 110 dB sound pressure level (SPL) of 16 kHz narrow-band noise (NBN) for 1 hour, and the restraint stress (RS) group was restrained for 1 hour with or without noise exposure. Gap prepulse inhibition of the acoustic startle (GPIAS) reflex was measured at an NBN of 16 kHz to investigate tinnitus development. Various immunohistopathologic and molecular biologic studies were undertaken to evaluate possible mechanisms of tinnitus development after noise and/or restraint stress., Results: The RS-only group showed a reduced GPIAS response, which is a reliable sign of tinnitus development. In the double-stimulus groups, more tinnitus-development signs of reduced GPIAS responses were observed. The expression of γ-aminobutyric acid A receptor α1 (GABAAR α1) in the hippocampus decreased in the NE│RS group. Increased N-methyl-d-aspartate receptor1 intensities in the NE│RS group and decreased GABAAR α1 intensities in the RS and NE│RS groups were observed in the CA3 region of the hippocampus., Conclusions: Tinnitus appeared to develop after stress alone in this animal study. An imbalance in excitatory and inhibitory neurotransmitters in the hippocampus may be related to the development of tinnitus after acute NE and/or stress., Level of Evidence: NA Laryngoscope, 131:2332-2340, 2021., (© 2021 The American Laryngological, Rhinological and Otological Society, Inc..)

Purpose: Most patients with metastatic melanoma show variable responses to radiation therapy and do not benefit from immune checkpoint inhibitors. Improved strategies for combination therapy that leverage potential benefits from radiation therapy and immune checkpoint inhibitors are critical., Methods and Materials: We analyzed metastatic melanoma tumors in the TCGA cohort for expression of genes coding for subunits of type A γ-aminobutyric acid (GABA) receptor (GABA A R), a chloride ion channel and major inhibitory neurotransmitter receptor. Electrophysiology was used to determine whether melanoma cells possess intrinsic GABA A R activity. Melanoma cell viability studies were conducted to test whether enhancing GABA A R mediated chloride transport using benzodiazepine-impaired viability. A syngeneic melanoma mouse model was used to assay the effect of benzodiazepine on tumor volume and its ability to potentiate radiation therapy or immunotherapy. Treated tumors were analyzed for changes in gene expression by RNA sequencing and presence of tumor-infiltrating lymphocytes by flow cytometry., Results: Genes coding for subunits of GABA A Rs express functional GABA A Rs in melanoma cells. By enhancing GABA A R-mediated anion transport, benzodiazepines depolarize melanoma cells and impair their viability. In vivo, benzodiazepine alone reduces tumor growth and potentiates radiation therapy and α-PD-L1 antitumor activity. The combination of benzodiazepine, radiation therapy, and α-PD-L1 results in near complete regression of treated tumors and a potent abscopal effect, mediated by increased infiltration of polyfunctional CD8 + T cells. Treated tumors show expression of cytokine-cytokine receptor interactions and overrepresentation of p53 signaling., Conclusions: This study identifies an antitumor strategy combining radiation and/or an immune checkpoint inhibitor with modulation of GABA A Rs in melanoma using benzodiazepine., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The GABA-A receptor signaling pathway regulates proliferation, differentiation, apoptosis, and responses to overt DNA damage during embryonic development., Methods: To analyze the gene expression after intracytoplasmic sperm injection (ICSI) and in in vivo mouse embryos, the global pattern of gene expression dataset, GSE23009, was obtained from the Gene Expression Omnibus database. Genes with differential expression were identified using the R software package, and RT-qPCR was performed to confirm the microarray results., Results: Mouse blastocysts derived from ICSI fertilization had decreased expression of GABA-A receptor signaling pathway genes. However, the mechanisms underlying these changes were not elucidated. The gene expression of the GABA-A pathway was not significantly different between blastocysts obtained from IVF and in vivo fertilization. However, microinjection after IVF significantly reduced the expression of the GABA-A pathway gene to levels similar to those in the ICSI group., Conclusion: Based on our results, decreased gene expression is a result of the microinjection manipulation performed during ICSI., (© 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

GABA A receptors are composed of five subunits arranged around a central chloride channel. Their subunits originate from different genes or gene families. The majority of GABA A receptors in the mammalian brain consist of two α-, two β- and one γ- or δ-subunit. This subunit organization crucially determines the physiological and pharmacological properties of the GABA A receptors. Using immunohistochemistry, we investigated the distribution of 10 GABA A receptor subunits (α1, α2, α3, α4, α5, β1, β2, β3, γ2, and δ) in the fore brain of three female rhesus monkeys (Macaca mulatta). Within the cerebral cortex, subunits α1, α5, β2, β3, and γ2 were found in all layers, α2, α3, and β1 were more concentrated in the inner and outer layers. The caudate/putamen was rich in α1, α2, α5, all three β-subunits, γ2, and δ. Subunits α3 and α5 were more concentrated in the caudate than in the putamen. In contrast, α1, α2, β1, β2, γ2, and δ were highest in the pallidum. Most dorsal thalamic nuclei contained subunits α1, α2, α4, β2, β3, and γ2, whereas α1, α3, β1, and γ2 were most abundant in the reticular nucleus. Within the amygdala, subunits α1, α2, α5, β1, β3, γ2, and δ were concentrated in the cortical nucleus, whereas in the lateral and basolateral amygdala α1, α2, α5, β1, β3, and δ, and in the central amygdala α1, α2, β3, and γ2 were most abundant. Interestingly, subunit α3-IR outlined the intercalated nuclei of the amygdala. In the hippocampus, subunits α1, α2, α5, β2, β3, γ2, and δ were highly expressed in the dentate molecular layer, whereas α1, α2, α3, α5, β1, β2, β3, and γ2 were concentrated in sector CA1 and the subiculum. The distribution of GABA A receptor subunits in the rhesus monkey was highly heterogeneous indicating a high number of differently assembled receptors. In most areas investigated, notably in the striatum/pallidum, amygdaloid nuclei and in the hippocampus it was more diverse than in the rat and mouse indicating a more heterogeneous and less defined receptor assembly in the monkey than in rodent brain., (© 2020 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

Despite being a highly conserved protein, the precise role of the mitochondrial translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), remains elusive. The void created by studies that overturned a presumptive model that described TSPO/PBR as a mitochondrial cholesterol transporter for steroidogenesis has been filled with evidence that it can affect mitochondrial metabolic functions across different model systems. We previously reported that TSPO/PBR deficient steroidogenic cells upregulate mitochondrial fatty acid oxidation and presented a strong positive correlation between TSPO/PBR expression and tissues active in triglyceride metabolism or lipid storage. Nevertheless, the highlighting of inconsistencies in prior work has provoked reprisals that threaten to stifle progress. One frequent factoid presented as being supportive of a cholesterol import function is that there are no steroid-synthesizing cell types without high TSPO/PBR expression. In this study, we examine the hamster adrenal gland that is devoid of lipid droplets in the cortex and largely relies on de novo cholesterol biosynthesis and uptake for steroidogenesis. We find that Tspo expression in the hamster adrenal is imperceptible compared to the mouse. This observation is consistent with a substantially low expression of Cpt1a in the hamster adrenal, indicating minimal mitochondrial fatty acid oxidation capacity compared to the mouse. These findings provide further reinforcement that the much sought-after mechanism of TSPO/PBR function remains correlated with the extent of cellular triglyceride metabolism. Thus, TSPO/PBR could have a homeostatic function relevant only to steroidogenic systems that manage triglycerides associated with lipid droplets.

The flexibility of neuronal networks is believed to rely mainly on the plasticity of excitatory synapses. However, like their excitatory counterparts, inhibitory synapses also undergo several forms of synaptic plasticity. This review examines recent advances in the understanding of the molecular mechanisms leading to postsynaptic GABAergic plasticity. Specifically, modulation of GABAA receptor (GABAAR) number at postsynaptic sites plays a key role, with the interaction of GABAARs with the scaffold protein gephyrin and other postsynaptic scaffold/regulatory proteins having particular importance. Our understanding of these molecular interactions are progressing, based on recent insights into the processes of GABAAR lateral diffusion, gephyrin dynamics, and gephyrin nanoscale organization. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The critical roles played by GABA A receptors as inhibitory regulators of excitation in the central nervous system has been known for many years. Aberrant GABA A receptor function and trafficking deficits have also been associated with several diseases including anxiety, depression, epilepsy, and insomnia. As a consequence, important drug groups such as the benzodiazepines, barbiturates, and many general anesthetics have become established as modulators of GABA A receptor activity. Nevertheless, there is much we do not understand about the roles and mechanisms of GABA A receptors at neural network and systems levels. It is therefore crucial to develop novel technologies and especially chemical entities that can interrogate GABA A receptor function in the nervous system. Here, we describe the chemistry and characterization of a novel set of 4-PIOL and 4-PHP analogues synthesized with the aim of developing a toolkit of drugs that can photoinactivate GABA A receptors. Most of these new analogues show higher affinities/potencies compared with the respective lead compounds. This is indicative of cavernous areas being present near their binding sites that can be potentially associated with novel receptor interactions. The 4-PHP azide-analogue, 2d , possesses particularly impressive nanomolar affinity/potency and is an effective UV-inducible photoinhibitor of GABA A receptors with considerable potential for photocontrol of GABA A receptor function in situ .

Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.

The striatum is the main input nucleus of the basal ganglia, mediating motor and cognitive functions. Striatal projection neurons are GABAergic medium spiny neurons (MSN), expressing either the dopamine receptor type 1 (D 1 -R MSN) and forming the direct, movement-promoting pathway, or dopamine receptor type 2 (D 2 -R MSN), forming the indirect movement-suppressing pathway. Locally, activity and synchronization of MSN are modulated by several subtypes of GABAergic and cholinergic interneurons. Overall, GABAergic circuits in the striatum remain poorly characterized, and little is known about the intrastriatal connectivity of interneurons and the distribution of GABA A receptor (GABA A R) subtypes, distinguished by their subunit composition, in striatal synapses. Here, by using immunofluorescence in mouse tissue, we investigated the distribution of GABA A Rs containing the α 1 , α 2 , or α 3 subunit in perisomatic synapses of striatal MSN and interneurons, as well as the innervation pattern of D 1 R- and D 2 R-MSN soma and axonal initial segment (AIS) by GABAergic and cholinergic interneurons. Our results show that perisomatic GABAergic synapses of D 1 R- and D 2 R-MSN contain the GABA A R α 1 and/or α 2 subunits, but not the α 3 subunit; D 2 R-MSN have significantly more α 1 -GABA A Rs on their soma than D 1 R-MSN. Further, interneurons have few perisomatic synapses containing α 2 -GABA A Rs, whereas α 3 -GABA A Rs (along with the α 1 -GABA A Rs) are abundant in perisomatic synapses of CCK + , NPY + /SOM + , and vAChT + interneurons. Each MSN and interneuron population analyzed received a distinct pattern of GABAergic and cholinergic innervation, complementing this postsynaptic heterogeneity. In conclusion, intra-striatal GABAergic circuits are distinguished by cell-type specific innervation patterns, differential expression and postsynaptic targeting of GABA A R subtypes., (© 2019 Wiley Periodicals, Inc.)

Presynaptic α 2 δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α 2 δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α 2 δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABA A Rs. Strikingly, presynaptic α 2 δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABA A Rs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α 2 δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α 2 δ-2 can modulate postsynaptic GABA A R abundance. Finally, exclusion of the alternatively spliced exon 23 of α 2 δ-2 is essential for the trans-synaptic mechanism. The novel function of α 2 δ-2 identified here may explain how abnormal α 2 δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders. SIGNIFICANCE STATEMENT Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α 2 δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α 2 δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α 2 δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia., (Copyright © 2019 Geisler et al.)

Rheumatoid arthritis (RA) is a chronic disease with systemic inflammation resulting in destruction of multiple articular cartilages and bones. Activated macrophage plays a pivotal role during the disease course and has been one of main targets to inhibit inflammatory reaction of RA by using biological disease-modifying anti-rheumatic drugs (bDMARDs). 18 F-FEDAC is one of PET imaging agents targeting TSPO, which is overexpressed in activated macrophages. The aim of this study was to evaluate the roles of 18 F-FEDAC PET as an in vivo imaging of activated macrophages on etanercept (ETN), a TNF-antagonist as one of bDMARDs in collagen induced arthritis mice. In RAW 264.7 cells, the expressions of TSPO as well as iNOS and infiltrated nucleus of NF-κB were induced by activation with lipopolysaccharide and interferon-gamma. TSPO expression was slightly attenuated by ETN treatment, not by methotrexate (MTX) as a cytotoxic agent. However, cell uptake of 18 F-FEDAC did not show significant changes according to both of the treatments. Similarly in CIA mice, 18 F-FEDAC uptake in inflamed paws on PET imaging did not show significant changes during both of the treatments, contrary to the uptake decrease of 18 F-FDG, a glucose analog to reflect metabolic or active inflammatory activity. Interestingly, when we divided joints according to the degree of 18 F-FEDAC uptake before ETN treatment, the joints of high 18 F-FEDAC uptake showed better response to ETN than the joints with low 18 F-FEDAC uptakes. In case of 18 F-FDG, there was no such kinds of patterns. We can speculate that 18 F-FEDAC PET imaging may identify activated macrophage-induced arthritis because that 18 F-FEDAC can reflect activated macrophages, which is the therapeutic target of ETN by TNF antagonistic effect. Thus, in vivo imaging using 18 F-FEDAC may be used as a predictor of therapeutic effects among those kinds of bDMARDs having anti-inflammatory actions to inhibit activated macrophage., (Copyright © 2018 Elsevier Inc. All rights reserved.)

ABSTRACTBackground:Although recent studies have suggested that the γ-aminobutyric acid type A (GABAA) receptor binding affinity can be a more sensitive marker of age-related neuronal loss than regional gray matter (GM) volume, knowledge about the relationship between decreased GABAA receptor binding affinity and cognitive decline during normal aging is still limited., Methods: Thirty-seven healthy elderly individuals (aged 50-77 years (mean, 64.5 ± 7.3 years); 15 males and 22 females) were enrolled in this study. We investigated the association of the performance of the healthy elderly in the attentional function test with regional GM volume, regional cerebral bold flow (rCBF), and GABAA receptor binding affinity in the resting state by structural magnetic resonance imaging (MRI), arterial spin labeling (ASL), and 123I-iomazenil (IMZ) SPECT, with the analysis focusing on the bilateral inferior frontal gyri., Results: The score of the rapid visual information processing (RVP) test, which is used to assess visual sustained attention, showed a positive correlation with GABAA receptor binding affinity in the right inferior frontal gyrus. No significant correlation was found between RVP test score and regional GM volume or rCBF., Conclusion: The findings of 123I-IMZ SPECT, but not those of structural MRI or ASL, suggest that a decreased GABAA receptor binding affinity can be a sensitive marker of cognitive impairment.

Cell surface expression of type A GABA receptors (GABA A Rs) is a critical determinant of the efficacy of inhibitory neurotransmission. Pentameric GABA A Rs are assembled from a large pool of subunits according to precise co-assembly rules that limit the extent of receptor structural diversity. These rules ensure that particular subunits, such as ρ1 and β3, form functional cell surface ion channels when expressed alone in heterologous systems, whereas other brain-abundant subunits, such as α and γ, are retained within intracellular compartments. Why some of the most abundant GABA A R subunits fail to form homomeric ion channels is unknown. Normally, surface expression of α and γ subunits requires co-assembly with β subunits via interactions between their N-terminal sequences in the endoplasmic reticulum. Here, using molecular biology, imaging, and electrophysiology with GABA A R chimeras, we have identified two critical residues in the transmembrane domains of α and γ subunits, which, when substituted for their ρ1 counterparts, permit cell surface expression as homomers. Consistent with this, substitution of the ρ1 transmembrane residues for the α subunit equivalents reduced surface expression and altered channel gating, highlighting their importance for GABA A R trafficking and signaling. Although not ligand-gated, the formation of α and γ homomeric ion channels at the cell surface was revealed by incorporating a mutation that imparts the functional signature of spontaneous channel activity. Our study identifies two single transmembrane residues that enable homomeric GABA A R subunit cell surface trafficking and demonstrates that α and γ subunits can form functional ion channels., (© 2018 Hannan and Smart.)

Potassium-chloride cotransporter 2 (KCC2) has been indicated to serve a crucial role during chronic neuropathic pain (NP). Following the emergence of NP, γ‑aminobutyric acid (GABA) A receptor‑mediated signaling may be further impaired by the changes of KCC2 chloride anion gradient. In the present study, the authors investigate the effect of electro-acupuncture (EA) on the behavior and the expression of KCC2 and GABAA receptor γ2 subunit in the spinal cord of chronic constriction injury (CCI) model rats. A total of 60 adult male Sprague‑Dawley rats were divided into four groups: Normal group, sham‑CCI group, CCI group and CCI+EA group. The effect of EA was assessed via the values of mechanical withdrawal threshold and thermal withdrawal latency, which were significantly improved upon stimulation of the ST‑36 and GB‑34 acupoints. In addition, a marked reduction in both the mRNA and protein levels of KCC2 and GABAA receptor γ2 subunit was observed in the spinal cord following loose ligation of the sciatic nerve. The reductions in KCC2 and GABAA receptor γ2 subunit expression were reversed by EA treatment. These results support the notion that KCC2 and GABAA receptor γ2 subunit contribute to NP following peripheral nerve injury and extend the understanding of the analgesic effects of EA on NP.

Numerous studies have reported age-dependent degradation of neuronal function in the visual cortex and have attributed this functional decline to weakened intracortical inhibition, especially GABAergic inhibition. However, whether this type of functional decline is linked to compromised GABAergic inhibition has not been fully confirmed. Here, we compared the neuronal response properties and markers of GABAergic inhibition in the primary visual cortex (V1) of young adult and senescent rats. Compared with those of young adult rats, old rats' V1 neurons exhibited significantly increased visually evoked responses and spontaneous activity, a decreased signal-to-noise ratio and reduced response selectivity for the stimulus orientation and motion direction. Additionally, the ratio of GABA-positive neurons to total cortical neurons in old rats was significantly decreased compared with that in young rats. Expression of the key GABA-synthesizing enzyme GAD67 was significantly lower in old rats than in young rats, although GAD65 expression showed a marginal difference between the two age groups. Further, expression of an important GABA A receptor subunit, GABA A R α 1 , was significantly attenuated in old rats relative to young ones. These results demonstrate that ageing may result in decreased GABAergic inhibition in the visual cortex and that this decrease in GABAergic inhibition accompanies neuronal function degradation.

The GABA A receptor is the most abundant inhibitory receptor in the human brain and is assembled from a variety of different subunit subtypes which determines their pharmacology and physiology. To determine which GABA A receptor subunit proteins are found in the human thalamus we investigated the distribution of five major GABA A receptor subunits α 1 , α 2 , α 3 , β 2,3 and γ 2 using immunohistochemical techniques. The α 1 -, β 2,3 - and γ 2 - subunits which combine to form a benzodiazepine sensitive GABA A receptor showed the most intense levels of staining and were the most common subunits found throughout the human thalamus especially in the ventral and posterior nuclear groups. The next most intense staining was for the α 3 -subunit followed by the α 2 -subunit. The intralaminar nuclear group, the mediodorsal nucleus and the thalamic reticular nucleus contained α 1 -, β 2,3 - and γ 2 - subunits staining as well as the highest levels of the α 2 - and α 3 - subunits. The sensory dorsal lateral geniculate nucleus contained very high levels of α 1 - and β 2,3 - and γ 2 -subunits. The highest densities of GABA A receptors found throughout the thalamus which contained the subunits α 1 , β 2,3 , and γ 2 included nuclei which are especially involved in the control or the modulation of the cortico-basal ganglia-thalamo-cortical motor circuits and are thus important in disorders such as Huntington's disease where the GABAergic projections of the basal ganglia are compromised. In addition the majority of receptors in the thalamic reticular nucleus contain α 3 and γ 2 subunits whilst the intralaminar nuclei contain high levels of α 2 and α 3 subunits., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background and Purpose: Misery perfusion may cause selective neuronal damage in atherosclerotic ICA or MCA disease. Bypass surgery can improve misery perfusion and may prevent neuronal damage. On the other hand, surgery conveys a risk for neuronal damage. The purpose of this retrospective study was to determine whether progression of cortical neuronal damage in surgically treated patients with misery perfusion is larger than that in surgically treated patients without misery perfusion or medically treated patients with misery perfusion., Materials and Methods: We evaluated the distribution of benzodiazepine receptors twice by using PET and 11 C-labeled flumazenil in 18 surgically treated patients with atherosclerotic ICA or MCA disease (9 with misery perfusion and 9 without) and no perioperative stroke before and after bypass surgery; in 8 medically treated patients with misery perfusion and no intervening ischemic event; and in 7 healthy controls. We quantified abnormal decreases in the benzodiazepine receptors of the cerebral cortex within the MCA distribution and compared changes in the benzodiazepine receptor index among the 3 groups., Results: The change in the benzodiazepine receptor index in surgically treated patients with misery perfusion (27.5 ± 15.6) during 7 ± 5 months was significantly larger than that in surgically treated patients without misery perfusion (-5.2 ± 9.4) during 6 ± 4 months ( P < .001) and in medically treated patients with misery perfusion (3.2 ± 15.4) during 16 ± 6 months ( P < .01)., Conclusions: Progression of cortical neuronal damage in surgically treated patients with misery perfusion and no perioperative stroke may occur and may be larger than that in medically treated patients with misery perfusion and no intervening ischemic event., (© 2017 by American Journal of Neuroradiology.)

Classical benzodiazepines, which are widely used as sedatives, anxiolytics and anticonvulsants, exert their therapeutic effects through interactions with heteropentameric GABA A receptors composed of two α, two β and one γ2 subunit. Their high affinity binding site is located at the interface between the γ2 and the adjacent α subunit. The α-subunit gene family consists of six members and receptors can be homomeric or mixed with respect to the α-subunits. Previous work has suggested that benzodiazepine binding site ligands with selectivity for individual GABA A receptor subtypes, as defined by the benzodiazepine-binding α subunit, may have fewer side effects and may even be effective in diseases, such as schizophrenia, autism or chronic pain, that do not respond well to classical benzodiazepines. The distributions of the individual α subunits across the CNS have been extensively characterized. However, as GABA A receptors may contain two different α subunits, the distribution of the subunits does not necessarily reflect the distribution of receptor subtypes with respect to benzodiazepine pharmacology. In the present study, we have used in vivo [ 18 F]flumazenil PET and in vitro [ 3 H]flumazenil autoradiography in combination with GABA A receptor point-mutated mice to characterize the distribution of the two most prevalent GABA A receptor subtypes (α1 and α2) throughout the mouse brain. The results were in agreement with published in vitro data. High levels of α2-containing receptors were found in brain regions of the neuronal network of anxiety. The α1/α2 subunit combinations were predictable from the individual subunit levels. In additional experiments, we explored in vivo [ 18 F]flumazenil PET to determine the degree of receptor occupancy at GABA A receptor subtypes following oral administration of diazepam. The dose to occupy 50% of sensitive receptors, independent of the receptor subtype(s), was 1-2mg/kg, in agreement with published data from ex vivo studies with wild type mice. In conclusion, we have resolved the quantitative distribution of α1- and α2-containing homomeric and mixed GABA A receptors in vivo at the millimeter scale and demonstrate that the regional drug receptor occupancy in vivo at these GABA A receptor subtypes can be determined by [ 18 F]flumazenil PET. Such information should be valuable for drug development programs aiming for subtype-selective benzodiazepine site ligands for new therapeutic indications., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Protein-based fluorescent biosensors are powerful tools for quantitative detection of biomolecules or drugs with high sensitivity under physiological conditions. However, conventional methods for construction of biosensors require structural data with high resolution or amino acid sequence information in most cases, which hampers applicability of this method to structurally complicated receptor proteins. To sidestep such limitations, we recently developed a new method that employs ligand-directed chemistry coupled with a bimolecular fluorescence quenching and recovery system, which enabled the conversion of various kinds of membrane-bound receptors to "turn-on" type fluorescent sensors. Here, we describe a protocol for construction of "turn-on" type fluorescent biosensors based on the GABA A receptor which permits quantitative analysis of the ligand affinity., (© 2017 Elsevier Inc. All rights reserved.)

Studies in rodents suggest that flumazenil is a P-glycoprotein substrate at the blood-brain barrier. This study aimed to assess whether [ 11 C]flumazenil is a P-glycoprotein substrate in humans and to what extent increased P-glycoprotein function in epilepsy may confound interpretation of clinical [ 11 C]flumazenil studies used to assess gamma-aminobutyric acid A receptors. Nine drug-resistant patients with epilepsy and mesial temporal sclerosis were scanned twice using [ 11 C]flumazenil before and after partial P-glycoprotein blockade with tariquidar. Volume of distribution, nondisplaceable binding potential, and the ratio of rate constants of [ 11 C]flumazenil transport across the blood-brain barrier (K 1 /k 2 ) were derived for whole brain and several regions. All parameters were compared between pre- and post-tariquidar scans. Regional results were compared between mesial temporal sclerosis and contralateral sides. Tariquidar significantly increased global K 1 /k 2 (+23%) and volume of distribution (+10%), but not nondisplaceable binding potential. At the mesial temporal sclerosis side volume of distribution and nondisplaceable binding potential were lower in hippocampus (both ∼-19%) and amygdala (both ∼-16%), but K 1 /k 2 did not differ, suggesting that only regional gamma-aminobutyric acid A receptor density is altered in epilepsy. In conclusion, although [ 11 C]flumazenil appears to be a (weak) P-glycoprotein substrate in humans, this does not seem to affect its role as a tracer for assessing gamma-aminobutyric acid A receptor density., (© The Author(s) 2015.)

Neuroinflammation is considered to be the pathogenesis of hepatic encephalopathy (HE), and imaging neuroinflammation is implicated in HE management. (11)C-PK11195, a typical translocator protein (TSPO) radiotracer, is used for imaging neuroinflammation. However, it has inherent limitations, such as short half-life and limited availability. The purpose of this study was to demonstrate the efficiency of new generation TSPO radiotracer, (18)F-DPA-714, in detecting and monitoring neuroinflammation of chronic HE. This study was divided into two parts. The first part compared (18)F-DPA-714 and (11)C-PK11195 radiotracers in ten HE induced rats [bile duct ligation (BDL) and fed hyperammonemic diet (HD)] and 6 control rats. The animal subjects underwent dynamic positron emission tomography (PET) during 2-day intervals. The (11)C-PK11195 PET study showed no differences in whole brain average percent injected dose per gram (%ID/g) values at all time points (all P>0.05), while the (18)F-DPA-714 PET study showed higher whole brain average %ID/g values in HE rats compared to control group rats at 900 s to 3300 s after injecting radiotracer (all P<0.05). The second part of the study evaluated the effectiveness of ibuprofen (IBU) treatment to chronic HE. Forty rats were classified into six groups, including Sham+normal saline (NS), Sham+IBU, BDL+NS, BDL+HD+NS, BDL+IBU, and BDL+HD+IBU groups. (18)F-DPA-714 PET was used to image neuroinflammation. Whole and regional brain average %ID/g values, neurological features, inflammatory factors and activated microglia showed better in the IBU groups than in the NS groups (all P<0.05) and no difference was seen in the Sham groups compared to IBU groups (all P>0.05). In conclusion, this study demonstrated that (18)F-DPA-714 is an ideal TPSO radiotracer for imaging neuroinflammation and monitoring anti-neuroinflammation treatment efficacy of chronic HE.

Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABA(A)R). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1-6, β1-3 or γ2 GABA(A)R subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABA(A)R subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABA(A)Rs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABA(A)R subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N = 16) and comparison (N = 14) subjects and found evidence of abnormal localization of the β1 and β2 GABA(A)R subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β2(52 kDa)), 50 kDa (β2(50 kDa)) and 48 kDa (β2(48 kDa)). In the ER, we found increased total β2 GABA(A)R subunit (β2(ALL)) expression driven by increased β2(50 kDa), a decreased ratio of β(248 kDa):β2(ALL) and an increased ratio of β2(50 kDa):β2(48 kDa). Decreased ratios of β1:β2(ALL) and β1:β2(50 kDa) in both the ER and SYN fractions and an increased ratio of β2(52 kDa):β(248 kDa) at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABA(A)Rs.

We evaluated the efficacy of 2-[5-(4-[(18)F]fluoroethoxy-2-oxo-1,3-benzoxazol-3(2H)-yl)-N-methyl-N-phenylacetamide] ([(18)F]FEBMP) for positron emission tomography (PET) imaging of translocator protein (18 kDa, TSPO). Dissection was used to determine the distribution of [(18)F]FEBMP in mice, while small-animal PET and metabolite analysis were used for a rat model of focal cerebral ischemia. [(18)F]FEBMP showed high radioactivity uptake in mouse peripheral organs enriched with TSPO, and relatively high initial brain uptake (2.67 ± 0.12% ID/g). PET imaging revealed an increased accumulation of radioactivity in the infarcted striatum, with a maximum ratio of 3.20 ± 0.12, compared to non-injured striatum. Displacement with specific TSPO ligands lowered the accumulation levels in infarcts to those on the contralateral side. This suggests that the increased accumulation reflected TPSO-specific binding of [(18)F]FEBMP in vivo. Using a simplified reference tissue model, the binding potential on the infarcted area was 2.72 ± 0.27. Metabolite analysis in brain tissues showed that 83.2 ± 7.4% and 76.4 ± 2.1% of radioactivity was from intact [(18)F]FEBMP at 30 and 60 min, respectively, and that this ratio was higher than in plasma (8.6 ± 1.9% and 3.9 ± 1.1%, respectively). In vitro autoradiography on postmortem human brains showed that TSPO rs6971 polymorphism did not affect binding sites for [(18)F]FEBMP. These findings suggest that [(18)F]FEBMP is a promising new tool for visualization of neuroinflammation.

The visualization of the activated microglia/TSPO is one of the main aspects of neuroimaging. Here we describe two new (18)F-labelled molecules, 2-[5-(4-[(18)F]fluoroethoxyphenyl)- ([(18)F]2) and 2-[5-(4-[(18)F]fluoropropyloxyphenyl)- ([(18)F]3) -2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-N-phenylacetamide as novel PET ligands for imaging the translocator protein (18 kDa, TSPO) in the brain. The three-D pharmacophore evaluation and docking studies suggested their high affinity for the TSPO and in vitro binding assays of the TSPO showed binding affinities 6.6 ± 0.7 nM and 16.7 ± 2.5 nM for 2 and 3, respectively. The radiochemical yields for [(18)F]2 and [(18)F]3 were found to be 22 ± 4% (n = 8) and 5 ± 2% (n = 5), respectively at EOB. The radiochemical purity for both was found ≥98% and the specific activity was in the range of 98-364 GBq μmol(-1) at EOS. In vitro autoradiography with an ischemic rat brain showed significantly increased binding on the ipsilateral side compared to the contralateral side. The specificity of [(18)F]2 and [(18)F]3 for binding TSPO was confirmed using the TSPO ligands PK11195 and MBMP. The biodistribution patterns of both PET ligands were evaluated in normal mice by 1 h dynamic PET imaging. In the brain, regional radioactivity reached the maximum very rapidly within 0-4 min for both ligands, similar to (R)[(11)C]PK11195. The metabolite study of [(18)F]2 also favoured a more favourable profile for quantification in comparison to (R)[(11)C]PK11195. In summary, these data indicated that [(18)F]2 and [(18)F]3 have good potential to work as PET ligands, therefore there are merits to use these radioligands for the in vivo evaluation in animal models to see their efficacy in the living brain.

Layer 1 of the neocortex harbors a unique group of neurons that play crucial roles in synaptic integration and information processing. Although extensive studies have characterized the properties of layer 1 neurons in the mature neocortex, it remains unclear how these neurons progressively acquire their distinct morphological, neurochemical, and physiological traits. In this study, we systematically examined the dynamic development of Cajal-Retzius cells and γ-aminobutyric acid (GABA)-ergic interneurons in layer 1 during the first 2 postnatal weeks. Cajal-Retzius cells underwent morphological degeneration after birth and gradually disappeared from layer 1. The majority of GABAergic interneurons showed clear expression of at least 1 of the 6 distinct neurochemical markers, including Reelin, GABA-A receptor subunit delta (GABAARδ), neuropeptide Y, vasoactive intestinal peptide (VIP), calretinin, and somatostatin from postnatal day 8. Furthermore, according to firing pattern, layer 1 interneurons can be divided into 2 groups: late-spiking (LS) and burst-spiking (BS) neurons. LS neurons preferentially expressed GABAARδ, whereas BS neurons preferentially expressed VIP. Interestingly, both LS and BS neurons exhibited a rapid electrophysiological and morphological development during the first postnatal week. Our results provide new insights into the molecular, morphological, and functional developments of the neurons in layer 1 of the neocortex., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The α4 subunit of the GABAA receptor (GABAAR) is highly expressed in the thalamus where receptors containing the α4 and δ subunits are major mediators of tonic inhibition. The α4 subunit also exhibits considerable plasticity in a number of physiological and pathological conditions, raising questions about the expression of remaining GABAAR subunits when the α4 subunit is absent. Immunohistochemical studies of an α4 subunit knockout (KO) mouse revealed a substantial decrease in δ subunit expression in the ventrobasal nucleus of the thalamus as well as other forebrain regions where the α4 subunit is normally expressed. In contrast, several subunits associated primarily with phasic inhibition, including the α1 and γ2 subunits, were moderately increased. Intracellular localization of the δ subunit was also altered. While δ subunit labeling was decreased within the neuropil, some labeling remained in the cell bodies of many neurons in the ventrobasal nucleus. Confocal microscopy demonstrated co-localization of this labeling with an endoplasmic reticulum marker, and electron microscopy demonstrated increased immunogold labeling near the endoplasmic reticulum in the α4 KO mouse. These results emphasize the strong partnership of the δ and α4 subunit in the thalamus and suggest that the α4 subunit of the GABAAR plays a critical role in trafficking of the δ subunit to the neuronal surface. The findings also suggest that previously observed reductions in tonic inhibition in the α4 subunit KO mouse are likely to be related to alterations in δ subunit expression, in addition to loss of the α4 subunit.

Anorexia nervosa (AN) is a psychiatric illness characterized by restricted eating and an intense fear of gaining weight. Most individuals with AN are females, diagnosed first during adolescence, 40-80% of whom exhibit excessive exercise, and an equally high number with a history of anxiety disorder. We sought to determine the cellular basis for individual differences in AN vulnerability by using an animal model, activity-based anorexia (ABA), that is induced by combining food restriction (FR) with access to a running wheel that allows voluntary exercise. Previously, we showed that by the fourth day of FR, the ABA group of adolescent female rats exhibit >500% greater levels of non-synaptic α4βδ-GABAARs at the plasma membrane of hippocampal CA1 pyramidal cell spines, relative to the levels found in age-matched controls that are not FR and without wheel access. Here, we show that the ABA group exhibits individual differences in body weight loss, with some losing nearly 30%, while others lose only 15%. The individual differences in weight loss are ascribable to individual differences in wheel activity that both precedes and concurs with days of FR. Moreover, the increase in activity during FR correlates strongly and negatively with α4βδ-GABAAR levels (R=-0.9, p<0.01). This negative correlation is evident within 2days of FR, before body weight loss approaches life-threatening levels for any individual. These findings suggest that increased shunting inhibition by α4βδ-GABAARs in spines of CA1 pyramidal neurons may participate in the protection against the ABA-inducing environmental factors of severe weight loss by suppressing excitability of the CA1 pyramidal neurons which, in turn, is related indirectly to suppression of excessive exercise. The data also indicate that, although exercise has many health benefits, it can be maladaptive to individuals with low levels of α4βδ-GABAARs in the CA1, particularly when combined with FR., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Introduction: Early diagnosis of and subsequent monitoring of therapy for rheumatoid arthritis (RA) could benefit from detection of (sub)clinical synovitis. Imaging of (sub)clinical arthritis by targeting the translocator protein (TSPO) on activated macrophages is feasible using (R)-[¹¹C] PK11195-based positron emission tomography (PET), but clinical applications are limited by background uptake in peri-articular bone/bone marrow. The purpose of the present study was to evaluate two other TSPO ligands with potentially lower background uptake in neurological studies, [¹¹C]DPA-713 and [¹⁸F]DPA-714, in a rat model of arthritis., Methods: TSPO binding of DPA-713, DPA-714 and PK11195 were assessed by in vitro competition studies with [³H]DPA-713 using human macrophage THP-1 cells and CD14⁺ monocytes from healthy volunteers. In vivo studies were performed in rats with methylated bovine serum albumin-induced knee arthritis. Immunohistochemistry with anti-TSPO antibody was performed on paraffin-embedded sections. Rats were imaged with [¹¹C]DPA-713 or [¹⁸F]DPA-714 PET, followed by ex vivo tissue distribution studies. Results were compared with those obtained with the tracer (R)-[¹¹C]PK11195, the established ligand for TSPO., Results: In THP-1 cells, relative TSPO binding of DPA-713 and DPA-714 were 7-fold and 25-fold higher, respectively, than in PK11195. Comparable results were observed in CD14⁺ monocytes from healthy volunteers. In the arthritis rat model, immunohistochemistry confirmed the presence of TSPO-positive inflammatory cells in the arthritic knee. PET images showed that uptake of [¹¹C]DPA-713 and [¹⁸F]DPA-714 in arthritic knees was significantly increased compared with contralateral knees and knees of normal rats. Uptake in arthritic knees could be largely blocked by an excess of PK11195. [¹¹C]DPA-713 and [¹⁸F]DPA-714 provided improved contrast compared with (R)-[¹¹C]PK11195, as was shown by significantly higher arthritic knee-to-bone ratios of [¹¹C]DPA-713 (1.60 ± 0.31) and [¹⁸F]DPA-714 (1.55 ± 0.10) compared with (R)-[¹¹C]PK11195 (1.14 ± 0.19)., Conclusions: [¹¹C]DPA-713 and [¹⁸F]DPA-714 clearly visualized arthritis and exhibited lower (peri-articular) bone/bone marrow uptake than (R)-[¹¹C]PK11195. These features merit further investigation of these tracers for early diagnosis and therapy monitoring of RA in a clinical setting.

Biochemical analysis of central nervous system proteins and nucleic acids requires fresh-tissue homogenates, whereas immunohistochemistry usually is performed in sections prepared from perfusion-fixed tissue. Post-mortem immersion-fixation is possible, but largely impairs morphological preservation and protein antigenicity. Here, we present a simple, fast and versatile protocol allowing concurrent biochemical and immunohistochemical analysis, including pre-embedding immunoelectron microscopy, using tissue from the same animal. The protocol includes a brief transcardiac perfusion with ice-cold, oxygenated and glucose-supplemented artificial cerebrospinal fluid to maintain brain tissue alive, prior to isolation of regions of interest, followed by homogenisation for biochemistry or immersion-fixation for immunohistochemistry. We provide several examples demonstrating that this protocol allows optimal biochemical and morphological analysis, characterised with optimal sensitivity and preservation of tissue structure, along with a reduction of artefacts typically seen in perfusion-fixed tissue. This protocol should find widespread applications for combining analytical methods in tissue from the same animal, thereby reducing the number of mice required for a given experiment., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Mitochondria represent an attractive subcellular target due to its function particularly important for oxidative damage, calcium metabolism and apoptosis. However, the concept of mitochondrial targeting has been a neglected area so far. The translocator protein (TSPO) represents an interesting subcellular target not only to image disease states overexpressing this protein, but also for a selective mitochondrial drug targeting. Recently, we have delivered in vitro and in vivo small molecule imaging agents into cells overexpressing TSPO by using a family of high-affinity conjugable ligands characterized by 2-phenyl-imidazo[1,2-a]pyridine acetamide structure. As an extension, in the present work we studied the possibility to target and image TSPO with dendrimers. These nano-platforms have unique features, in fact, are prepared with a level of control not reachable with most linear polymers, leading to nearly monodisperse, globular macromolecules with a large number of peripheral groups. As a consequence, they are an ideal delivery vehicle candidate for explicit study of the effects of polymer size, charge, composition, and architecture on biologically relevant properties such as lipid bilayer interactions, cytotoxicity, cellular internalization, and subcellular compartments and organelles interactions. Here, we present the synthesis, characterization, cellular internalization, and mitochondria labeling of a TSPO targeted fourth generation [G(4)-PAMAM] dendrimer nanoparticle labeled with the organic fluorescent dye fluorescein. We comprehensively studied the cellular uptake behavior of these dendrimers, into glioma C6 cell line, under the influence of various endocytosis inhibitors. We found that TSPO targeted-G(4)-PAMAM-FITC dendrimer is quickly taken up by these cells by endocytosis pathways, and moreover specifically targets the mitochondria as evidenced from subcellular fractionation experiments and co-localization studies performed with CAT (Confocal-AFM-TIRF) microscopy., (© 2013.)

Neuronal circuitries in the hippocampus are involved in navigation and memory and are controlled by major networks of GABAergic interneurons. Parvalbumin (PV)-expressing interneurons in the dentate gyrus (DG) are identified as fast-spiking cells, playing a crucial role in network oscillation and synchrony. The inhibitory modulation of these interneurons is thought to be mediated mainly through GABAA receptors, the major inhibitory neurotransmitter receptors in the brain. Here we show that all PV-positive interneurons in the granular/subgranular layer (GL/SGL) of the mouse DG express high levels of the GABAA receptor δ subunit. PV-containing interneurons in the hilus and the molecular layer, however, express the δ subunit to a lower extent. Only 8% of the somatostatin-containing interneurons express the δ subunit, whereas calbindin- or calretinin-containing interneurons in the DG seem not to express the GABAA receptor δ subunit at all. Hence, these cells receive a GABAergic control different from that of PV-containing interneurons in the GL/SGL. Experiments investigating a possible co-expression of GABAA receptor α1, α2, α3, α4, α5, β1, β2, β3, or γ2 subunits with PV and δ subunits indicated that α1 and β2 subunits are co-expressed with δ subunits along the extrasynaptic membranes of PV-interneurons. These results suggest a robust tonic GABAergic control of PV-containing interneurons in the GL/SGL of the DG via δ subunit-containing receptors. Our data are important for better understanding of the neuronal circuitries in the DG and the role of specific cell types under pathological conditions., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The GABAA receptor is the main inhibitory receptor in the brain and its subunits originate from different genes or gene families (α1-α6, β1-β3, γ1-γ3, δ, ε, θ, π, or ρ1-3). In the mouse brain the anatomical distribution of GABAA receptor subunit mRNAs so far investigated is restricted to subunits forming benzodiazepine-sensitive receptor complexes (α1-α3, α5, β2, β3 and γ2) in the forebrain and midbrain as assessed by in situ hybridization (ISH). In the present study the anatomical distribution of the GABAA receptor subunits α1-α6, β1-β3, γ1-γ2 and δ was analyzed in the mouse brain (excluding brain stem) by ISH and immunohistochemistry (IHC). In several brain areas such as hippocampus, cerebellum, bulbus olfactorius and habenula we observed that mRNA levels did not reflect protein levels, indicating that the protein is located far distantly from the cell body. We also compared the distribution of these 12 subunit mRNAs and proteins with that reported in the rat brain. Although in general there is a considerable correspondence in the distribution between mouse and rat brains, several species-specific differences were observed., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Ethanol modulates the GABA(A) receptor to cause sedative, anxiolytic and hypnotic effects that are qualitatively similar to benzodiazepines and barbiturates. The aim of this study was to explore if GABA(A) receptor density is altered in post-mortem brains of anxiety-prone Cloninger type 1 and socially hostile type 2 alcoholic subtypes when compared to controls. The GABA(A) binding site density was measured by whole-hemisphere autoradiography with tritium labeled flunitrazepam ([(3)H]flunitrazepam) from 17 alcoholic (nine type 1, eight type 2) and 10 non-alcoholic post-mortem brains, using cold flumazepam as a competitive ligand. A total of eight specific brain areas were examined. Alcoholics displayed a significantly (p < 0.001, bootstrap type generalizing estimating equations model) reduced [(3)H]flunitrazepam binding site density when compared to controls. When localized, type 2 alcoholics displayed a significantly (p ≤ 0.05) reduced [(3)H]flunitrazepam binding site density in the internal globus pallidus, the gyrus dentatus and the hippocampus, whereas type 1 alcoholics differed from controls in the internal globus pallidus and the hippocampus. While previous reports have demonstrated significant alterations in dopaminergic and serotonergic receptors between type 1 and type 2 alcoholics among these same subjects, we observed no statistically significant difference in [(3)H]flunitrazepam binding site densities between the Cloninger type 1 and type 2 alcoholics., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The accurate knowledge of receptor kinetics is crucial to our understanding of cell signal transduction in general and neural function in particular. The classical technique of probing membrane receptors on a millisecond scale involves placing a recording micropipette with a membrane patch in front of a double-barrel (θ-glass) application pipette mounted on a piezo actuator. Driven by electric pulses, the actuator can rapidly shift the θ-glass pipette tip, thus exposing the target receptors to alternating ligand solutions. However, membrane patches survive for only a few minutes, thus normally restricting such experiments to a single-application protocol. In order to overcome this deficiency, we have introduced pressurized supply microcircuits in the θ-glass channels, thus enabling repeated replacement of application solutions within 10-15 s. This protocol, which has been validated in our recent studies and takes 20-60 min to implement, allows the characterization of ligand-receptor interactions with high sensitivity, thereby also enabling a powerful paired-sample statistical design.

Objective: We examined regional benzodiazepine receptors (rBZR) using single photon emission CT (SPECT) in patients with Alzheimer disease (AD), vascular dementia (VaD), and mixed AD/VaD dementia (MD) and compared the changes in the availability of rBZR with those of regional cerebral blood flow (rCBF)., Methods: A total of 7 patients with AD, 6 with MD, and 9 with VaD underwent SPECT studies with N-isopropyl-p-[(123)I] iodoamphetamine and (123)I-iomazenil to measure rCBF and rBZR. The ratios of rCBF and rBZR uptake in brain subregions to the average global activity were compared among these diseases. In addition, we acquired z-score maps using 3-dimensional stereotactic surface projections of SPECT data., Results: Compared with AD, VaD and MD showed rCBF and rBZR reduction predominantly in the frontal lobe, but rBZR images revealed more extensive and severe defects than rCBF images. In contrast, AD showed rCBF and rBZR reduction predominantly in the parietotemporal lobe compared with VaD and MD, but rCBF images revealed more extensive defects than rBZR images., Conclusion: rCBF imaging can detect parietotemporal abnormalities in AD, while rBZR imaging may enable the demonstration of underlying pathophysiological differences in the frontal lobe between VaD, MD and AD, reflecting neuronal integrity in the cerebral cortex., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The acute and subacute ischemic neuronal damage in rat brain caused by photochemically induced thrombosis (PIT) was imaged using [¹⁸F]BMS-747158-02 ([¹⁸F]BMS) for mitochondrial complex-1 (MC-1) and [¹¹C](R)-PK11195 ([¹¹C](R)-PK) for peripheral benzodiazepine receptor [PBR; translocator protein] at preischemic "Normal," 1 (day 1), and 7 days (day 7) after ischemic insult. When [¹⁸F]BMS was intravenously injected into "Normal" rat, it was rapidly taken up into the brain, in which it showed a homogeneous distribution, and the uptake was suppressed by rotenone, a specific MC-1 inhibitor. The specificity of [¹⁸F]BMS binding to MC-1 was also confirmed by living brain slice imaging. At day 1, [¹⁸F]BMS uptake was low in infarct and peri-infarct regions where neuronal damage was detected by 2,3,5-triphenyltetrazolium chloride (TTC) staining. At day 7, the damaged areas determined using [¹⁸F]BMS revealed some discrepancy from those detected by TTC staining, suggesting that TTC stained not only surviving cells but also activated microglial cells in the peri-infarct region. This was also confirmed by [¹¹C](R)-PK imaging and immunohistochemical assessment with Iba1 antibody. In contrast, the uptake pattern of [¹⁸F]BMS was consistent with immunohistochemical assessment with NeuN antibody at both days 1 and 7. These results demonstrated that [¹⁸F]BMS could be a promising positron emission tomography ligand to assess the neuronal damage induced by ischemic insult in both acute and subacute phases., (Copyright © 2012 Wiley Periodicals, Inc.)

A series of novel ligands based on the diaryl anilide (DAA) class of translocator protein (TSPO) ligands was synthesised and evaluated as potential positron emitting tomography (PET) ligands for imaging TPSO in vivo. Fluorine-18 labelling of the molecules was achieved using direct radiolabelling or synthon based labelling approaches. Several of the ligands prepared have promising profiles as potential TSPO PET imaging ligands and will be evaluated further as potential clinical imaging agents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Asthma is a chronic inflammatory disease characterized by reversible bronchial constriction, pulmonary inflammation and airway remodeling. Current standard therapies for asthma provide symptomatic control, but fail to target the underlying disease pathology. Furthermore, no therapeutic agent is effective in preventing airway remodeling. A substantial amount of evidence suggests that statins have anti-inflammatory properties and immunomodulatory activity. In this study, we investigated the effect of rosuvastatin on airway inflammation and its inhibitory mechanism in mucus hypersecretion in a murine model of chronic asthma., Methods: BALB/c mice were sensitized and challenged by ovalbumin to induce asthma. The recruitment of inflammatory cells into bronchoalveolar lavage fluid (BALF) and the lung tissues were measured by Diff-Quik staining and hematoxylin and eosin (H&E) staining. ELISA was used for measuring the levels of IL-4, IL-5, IL-13 and TNF-α in BALF. Periodic acid-Schiff (PAS) staining was used for mucus secretion. Gamma-aminobutyric acid type A receptor (GABAAR) β2 expression was measured by means of immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting., Results: Rosuvastatin reduced the number of total inflammatory cells, lymphocytes, macrophages, neutrophils, and eosinophils recruited into BALF, the levels of IL-4, IL-5, IL-13 and TNF-α in BALF, along with the histological mucus index (HMI) and GABAAR β2 expression. Changes occurred in a dose-dependent manner., Conclusions: Based on its ability to reduce the inflammatory response and mucus hypersecretion by regulating GABAAR activity in a murine model of chronic asthma, rosuvastatin may be a useful therapeutic agent for treatment of asthma.

Phospholipase C-related, but catalytically inactive protein (PRIP) was first identified as a novel inositol 1,4,5-triphosphate binding protein. The PRIP-1 subtype is expressed predominantly in the central nervous system and binds directly to the GABA type A receptor (GABA(A)-R) β-subunit and several other proteins involved in the trafficking of GABA(A)-Rs to the plasma membrane. We found that the PRIP-1 knockout mouse showed an epileptic phenotype, confirmed by electroencephalogram. These ictal seizures were completely suppressed by diazepam (DZP), but the interictal discharges could not be abolished. We studied the electrophysiological properties of GABAergic transmission in hippocampal CA1 pyramidal neurons, using a slice patch-clamp technique. There was no difference in the effect of up to 1 μM DZP on the amplitude and frequency of miniature inhibitory postsynaptic currents between PRIP-1 knockout neurons versus wild-type neurons. In contrast, the amplitude of the tonic GABA current in PRIP-1 knockout neurons was markedly reduced compared with that in wild-type neurons. Consequently, the effect of DZP on PRIP-1 knockout mice was reduced. Dysfunction of extrasynaptic GABAergic transmission probably is involved in the epileptic phenotype of PRIP-1 knockout mice.

In atherosclerotic internal carotid artery (ICA) or middle cerebral artery (MCA) disease, selective neuronal damage can be detected as a decrease in central benzodiazepine receptors (BZRs) in an apparently normal cerebral cortex. To investigate the association between cortical BZRs decreases and executive dysfunctions, we measured BZRs using positron emission tomography (PET) and (11)C-flumazenil in 60 non-disabled patients with unilateral atherosclerotic ICA or MCA disease and no cortical infarction. Using 3-dimensional stereotactic surface projections, we calculated the BZR index, a measure of abnormally decreased BZRs in the cerebral cortex within the anterior cerebral artery (ACA) or MCA territory, and found that it to be correlated with the patient's score on the Wisconsin Card Sorting Test (WCST). Based on the WCST results, 39 patients were considered abnormal (low categories achieved) for their age. The BZR index of the ACA territory in the hemisphere affected by arterial disease was significantly higher in abnormal patients than in normal patients. The BZR index of the MCA territory differed significantly between the 2 groups when patients with left arterial disease (n = 28) were analyzed separately. The BZR indices of the anterior cingulate gyrus and the middle frontal gyrus carrelated significantly and positively with the total number of WCST errors. In atherosclerotic ICA or MCA disease, selective neuronal damage that is manifested as a decrease in BZRs in the non-infarcted cerebral cortex is associated with executive dysfunction. PET imaging of BZRs is useful as an objective measure of cognitive impairments in atherosclerotic occlusive disease of the major cerebral artery.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABA(A) and metabotropic GABA(B) receptors. During the development of the nervous system, GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation, and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABA(A) and GABA(B) receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABA(A) α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern, suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner., (Copyright © 2011 Wiley-Liss, Inc.)

Mice overexpressing CB2r (CB2xP) were exposed to open field (OF), light-dark box (LDB) and elevated plus maze (EPM) tests. Corticotropin-releasing factor (CRF) and pro-opiomelanocortin (POMC) mRNA were measured in paraventricular (PVN) and arcuate (ARC) nuclei of the hypothalamus after 30 minutes of restraint stress (RS). Anxiolytic effects of alprazolam (45 or 70 µg/kg, ip) were evaluated. GABA(A)α(2) and GABA(A)γ(2) mRNA were measured in the hippocampus (HIPP) and amygdala (AMY) of CB2xP and wild type (WT) mice. No differences were observed in the total distance travelled by CB2xP and WT mice in OF. Central and peripheral distances travelled significantly increased and decreased in CB2xP mice. Overexpression of CB2r reduced anxiety-like behaviours in LDB and EPM. In WT mice, RS increased CRF (82%) and POMC (42%) mRNA in the PVN and ARC nuclei, respectively. In CB2xP mice, RS also increased POMC (22%) mRNA in the ARC nucleus, but had no effect on CRF mRNA in the PVN nucleus. Administration of alprazolam was without effect in CB2xP mice. An increase of GABA(A)α(2) and GABA(A)γ(2) mRNA in the hippocampus and amygdala of CB2xP mice was observed. Our findings revealed that increased expression of CB2r significantly reduced anxiogenic-related behaviours, modified the response to stress and impaired the action of anxiolytic drugs.

Unlabelled: Translocator protein (TSPO), also referred to as peripheral benzodiazepine receptor (PBR), is a crucial 18-kDa outer mitochondrial membrane protein involved in numerous cellular functions, including the regulation of cholesterol metabolism, steroidogenesis, and apoptosis. Elevated expression of TSPO in oncology correlates with disease progression and poor survival, suggesting that molecular probes capable of assaying TSPO levels may have potential as cancer imaging biomarkers. In preclinical PET studies, we characterized a high-affinity aryloxyanilide-based TSPO imaging ligand, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (18F-PBR06), as a candidate probe for the quantitative assessment of TSPO expression in glioma., Methods: Glioma-bearing rats were imaged with 18F-PBR06 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of 18F-PBR06 (70-100 MBq/0.2 mL). Over the course of scanning, arterial blood was collected to derive the input function, with high-performance liquid chromatography radiometabolite analysis performed on selected samples for arterial input function correction. Compartmental modeling of the PET data was performed using the corrected arterial input function. Specific tumor cell binding of PBR06 was evaluated by radioligand displacement of 3H-PK 11195 with PBR06 in vitro and by displacement of 18F-PBR06 with excess PBR06 in vivo. Immediately after imaging, tumor tissue and adjacent healthy brain were harvested for assay of TSPO protein levels by Western blotting and immunohistochemistry., Results: 18F-PBR06 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain, facilitating excellent contrast between tumor and adjacent tissue. Infusion with PBR06 (10 mg/kg) displaced 18F-PBR06 binding by approximately 75%. The accumulation of 18F-PBR06 in tumor tissues and adjacent brain agreed with the ex vivo assay of TSPO protein levels by Western blotting and quantitative immunohistochemistry., Conclusion: These preclinical studies illustrate that 18F-PBR06 is a promising tracer for visualization of TSPO-expressing tumors. Importantly, the close correlation between 18F-PBR06 uptake and TSPO expression in tumors and normal tissues, coupled with the high degree of displaceable binding from both tumors and the normal brain, represents a significant improvement over other TSPO imaging ligands previously evaluated in glioma. These data suggest the potential of 18F-PBR06 to elucidate the role of TSPO in oncology, as well as its potential development as a cancer imaging biomarker.

The principal function of the γ-aminobutyric acid (GABA) system in the adult brain is inhibition; however, in the neonatal brain, GABA provides much of the excitatory drive. As the brain develops, transmembrane chloride gradients change and the inhibitory role of GABA is initiated and continues throughout juvenile and adult life. Previous studies have shown that GABA(A) receptor subunit expression is developmentally regulated, and it is thought that the change in GABA function from excitation to inhibition corresponds to the changeover in expression of 'immature' to 'mature' subunit isoforms. We examined the protein expression pattern and distribution of GABA type A (GABA(A)) receptor α1-, α3- and β2-subunits in the parietal cortex and hippocampus of the developing piglet brain. Four perinatal ages were studied; 14 days preterm (P-14), 10 days preterm (P-10), day of birth (P0) and at postnatal day 7 (P7). Animals were obtained by either caesarean section or spontaneous birth. Protein expression levels and subunit localization were analysed by Western blotting and immunohistochemistry, respectively. In the cortex and hippocampus, GABA(A) receptor α1-subunit showed greatest expression at P7 when compared to all other age groups (p < 0.05). In contrast, α3 expression in the cortex was elevated in preterm brain, peaking at P0, followed by a significant reduction by P7 (p < 0.05); a similar trend was observed in the hippocampus. GABA(A) receptor β2-subunit protein expression appeared relatively constant across all time points studied in both the cortex and hippocampus. Immunolabelling of the α1-, α3- and β2-subunits was observed throughout all cortical layers at every age. GABA(A) receptor α3-subunit appeared to show specific localization to layers V and VI whilst labelling for the β2-subunit was observed in layer IV. In the hippocampus of all animals, the α1- and β2-subunits were shown to immunolabel various cells and processes in the dentate gyrus (DG), CA1 and CA3; the α3-subunit was barely observed except at the stratum moleculare of the DG. We report for the first time the ontogenesis of GABA(A) receptor subunits α1, α3 and β2 in the perinatal pig brain., (Copyright © 2011 S. Karger AG, Basel.)

We evaluated cortical damages following traumatic brain injury (TBI) in the acute phase with [(123)I] iomazenil (IMZ) single photon emission computed tomography (SPECT). In all, 12 patients with cerebral contusion following TBI were recruited. All patients underwent IMZ SPECT within 1 week after TBI. To investigate the changes in distribution of IMZ in the cortex in the chronic phase, after conventional treatment, patients underwent IMZ SPECT again. A decrease in the accumulation of radioligand for the central benzodiazepine receptor in the cortex corresponding to the contusion revealed with computed tomography (CT) scans and magnetic resonance imaging (MRI) were shown on IMZ SPECT in the acute phase in all patients. In 9 of 12 patients (75%), images of IMZ SPECT obtained in the chronic phase of TBI showed that areas with a decreased distribution of IMZ were remarkably reduced in comparison with those obtained in the acute phase. Both CT scans and MRI showed a normal appearance of the cortex morphologically, where the binding potential of IMZ recovered in the chronic phase. Reduced binding potential of radioligand for the central benzodiazepine receptor is considered to be an irreversible reaction; however, in this study, IMZ accumulation in the cortex following TBI was recovered in the chronic phase in several patients. [(123)I] iomazenil SPECT may have a potential to disclose a reversible vulnerability of neurons following TBI.

Objective: To study the effect of maternal isolation stress on the epilepsy susceptibility in young rats and the possible mechanism., Methods: Sixty Sprague-Dawley young rats were randomly divided into a normal control and two maternal isolation groups that were subjected to maternal isolation for 15 min or 3 hrs daily on postnatal days 2-17. On postnatal day 18, an amygdala kindling test was performed to induce seizures. The expression of GABA(A) receptor α₁ in the hippocampus was determined by immunohistochemisty., Results: The weights were reduced, the threshold of amygdala kindling and the stimulation number for full kindling decreased significantly, and seizures were more severe in the maternal isolation 3 hrs group compared with the normal control group. The expression of GABA(A) receptor alpha(1) in the hippocampus CA1 area in the maternal isolation 3 hrs group decreased significantly compared with that in the normal groups. There were no significant differences in the aspects above mentioned between the maternal isolation 15 min and normal control groups., Conclusions: The stress of early daily maternal isolation for 3 hrs may affect adversely brain development and increase epilepsy susceptibility in young rats. The decreased expression of GABA(A) receptor α₁ in the hippocampus may contribute to the potential mechanism.

Neurosteroids such as allopregnanolone (THP) act as positive allosteric modulators of gamma-aminobutyric acid (GABA)A receptors and have exerted anticonvulsant properties. However, their role in the regulation of epileptogenesis is unclear. It has been shown that circulating levels of THP fluctuate during development and seizure episodes. Furthermore, both chronic administration of THP and its withdrawal transiently increase expression of the alpha4 subunit of the GABAA receptor in the brain. The steroidogenic enzymes, 5-alpha-reductase (5aR) and 3-alpha-hydroxysteroid dehydrogenase (3aHSD) have been identified as well, indicating that various cell types are involved in the biosynthesis of neuroactive steroids in the brain. The purpose of the present study is to examine how GABAA receptor-modulating neurosteroids contribute to the epileptogenesis by using the epileptic mutant EL mouse. Male EL mice and control animals, DDY mice, were used. EL mice show secondary generalized seizures, which initiate primarily at the parietal cortex and generalize through the hippocampus. In the interictal period during development, changes of THP, 5aR, 3aSDH, and GABAA receptor alpha4, gamma2, and delta subunits were investigated by western blotting in the hippocampus. In EL mice, levels of the neurosteroid THP and the steroidogenic enzymes 5aR and 3aSDH significantly increased at 3 weeks of age, and rapidly decreased thereafter (5-10 weeks). The sharp withdrawal was observed before mice experienced frequent seizures. In contrast, GABAA alpha4, gamma2, and delta expressions were upregulated (3-8 weeks). In the brain of EL mice, positive neurosteroids such as THP were withdrawn before mice experienced repetitive seizures, which may likely be a trigger for ictogenesis and epileptogenesis. Furthermore, reorganization of the GABAA receptor subunits may lead to a hypersensitivity of the receptor to neurosteroids. Therefore, GABAA receptor-regulating neurosteroids may be a promising target for the development of novel antiepileptic agents.

We have shown altered expression of gamma-aminobutyric acid A (GABA(A)) and gamma-aminobutyric acid B (GABA(B)) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3 GABA(A) subunits previously associated with autism (GABRalpha4; GABRalpha5; GABRbeta1). Three GABA receptor subunits demonstrated mRNA and protein level concordance in superior frontal cortex (GABRalpha4, GABRalpha5, GABRbeta1) and one demonstrated concordance in cerebellum (GABBetaR1). These results provide further evidence of impairment of GABAergic signaling in autism.

The gabaergic system has been identified as a relevant regulator of cognitive and emotional processing. In fact, the discovery that negative allosteric regulators (or inverse agonists) at GABA(A) (γ-aminobutyric acid) α5 subtype receptors improve learning and memory tasks, has further validated this concept. The localization of these extrasynaptic subtype receptors, mainly in the hippocampus, has suggested that they play a key role in the three stages of memory: acquisition, consolidation, and retrieval. The "α5 inverse agonist" binds to an allosteric site at GABA(A) receptor, provoking a reduction of chlorine current, but to elicit this effect, the necessary condition is the binding of agonist neurotransmitter (γ-amino butyric acid) at its orthosteric site. In this case, the GABA(A) receptor is not a "constitutively active receptor" and, however, the presence of spontaneous opening channels for native GABA(A) receptors is rare. Here, we present various classes of nonselective and α5 selective GABA(A) receptor ligands, and the in vitro and in vivo tests to elucidate their affinity and activity. The study of the GABA(A) α5 inverse agonists is one of the important tools, although not the only one, for the development of clinical strategies for treatment of Alzheimer disease and mild cognitive impairment., (Copyright © 2010 Elsevier Inc. All rights reserved.)