Translational evaluation of translocator protein as a marker of neuroinflammation in schizophrenia.

Positron emission tomography (PET) imaging with radiotracers that target translocator protein 18 kDa (TSPO) has become a popular approach to assess putative neuroinflammatory processes and associated microglia activation in psychotic illnesses. It remains unclear, however, whether TSPO imaging can accurately capture low-grade inflammatory processes such as those present in schizophrenia and related disorders. Therefore, we evaluated the validity of TSPO as a disease-relevant marker of inflammation using a translational approach, which combined neurodevelopmental and neurodegenerative mouse models with PET imaging in patients with recent-onset schizophrenia and matched controls. Using an infection-mediated neurodevelopmental mouse model, we show that schizophrenia-relevant behavioral abnormalities and increased inflammatory cytokine expression are associated with reduced prefrontal TSPO levels. On the other hand, TSPO was markedly upregulated in a mouse model of acute neurodegeneration and reactive gliosis, which was induced by intrahippocampal injection of kainic acid. In both models, the changes in TSPO levels were not restricted to microglia but emerged in various cell types, including microglia, astrocytes and vascular endothelial cells. Human PET imaging using the second-generation TSPO radiotracer [11C]DPA-713 revealed a strong trend towards reduced TSPO binding in the middle frontal gyrus of patients with recent-onset schizophrenia, who were previously shown to display increased levels of inflammatory cytokines in peripheral and central tissues. Together, our findings challenge the common assumption that central low-grade inflammation in schizophrenia is mirrored by increased TSPO expression or ligand binding. Our study further underscores the need to interpret altered TSPO binding in schizophrenia with caution, especially when measures of TSPO are not complemented with other markers of inflammation. Unless more selective microglial markers are available for PET imaging, quantification of cytokines and other inflammatory biomarkers, along with their molecular signaling pathways, may be more accurate in attempts to characterize inflammatory profiles in schizophrenia and other mental disorders that lack robust reactive gliosis.

Molecular and neuronal substrate for the selective attenuation of anxiety.

Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and analyzed two mouse lines in which the alpha2 or alpha3 GABAA (gamma-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the alpha2(H101R) point mutation but present in mice with the alpha3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by alpha2 GABAA receptors, which are largely expressed in the limbic system, but not by alpha3 GABAA receptors, which predominate in the reticular activating system.

Adjacent phosphorylation sites on GABAA receptor beta subunits determine regulation by cAMP-dependent protein kinase.

Activation of cAMP-dependent protein kinase (PKA) can enhance or reduce the function of neuronal GABAA receptors, the major sites of fast synaptic inhibition in the brain. This differential regulation depends on PKA-induced phosphorylation of adjacent conserved sites in the receptor beta subunits. Phosphorylation of beta 3 subunit-containing receptors at S408 and S409 enhanced the GABA-activated response, whereas selectively mutating S408 to alanine converted the potentiation into an inhibition, comparable to that of beta 1 subunits, which are phosphorylated solely on S409. These distinct modes of regulation were interconvertible between beta 1 and beta 3 subunits and depended upon the presence of S408 in either subunit. In contrast, beta 2 subunit-containing receptors were not phosphorylated or affected by PKA. Differential regulation by PKA of postsynaptic GABAA receptors containing different beta subunits may have profound effects on neuronal excitability.

Decreased GABAA-receptor clustering results in enhanced anxiety and a bias for threat cues.

Patients with panic disorders show a deficit of GABAA receptors in the hippocampus, parahippocampus and orbitofrontal cortex. Synaptic clustering of GABAA receptors in mice heterozygous for the gamma2 subunit was reduced, mainly in hippocampus and cerebral cortex. The gamma2 +/- mice showed enhanced behavioral inhibition toward natural aversive stimuli and heightened responsiveness in trace fear conditioning and ambiguous cue discrimination learning. Implicit and spatial memory as well as long-term potentiation in hippocampus were unchanged. Thus gamma2 +/- mice represent a model of anxiety characterized by harm avoidance behavior and an explicit memory bias for threat cues, resulting in heightened sensitivity to negative associations. This model implicates GABAA-receptor dysfunction in patients as a causal predisposition to anxiety disorders.

Mastering hysteresis in magnetocaloric materials.

Hysteresis is more than just an interesting oddity that occurs in materials with a first-order transition. It is a real obstacle on the path from existing laboratory-scale prototypes of magnetic refrigerators towards commercialization of this potentially disruptive cooling technology. Indeed, the reversibility of the magnetocaloric effect, being essential for magnetic heat pumps, strongly depends on the width of the thermal hysteresis and, therefore, it is necessary to understand the mechanisms causing hysteresis and to find solutions to minimize losses associated with thermal hysteresis in order to maximize the efficiency of magnetic cooling devices. In this work, we discuss the fundamental aspects that can contribute to thermal hysteresis and the strategies that we are developing to at least partially overcome the hysteresis problem in some selected classes of magnetocaloric materials with large application potential. In doing so, we refer to the most relevant classes of magnetic refrigerants La-Fe-Si-, Heusler- and Fe2P-type compounds.This article is part of the themed issue 'Taking the temperature of phase transitions in cool materials'.

Genetic inactivation of the Serotonin(1A) receptor in mice results in downregulation of major GABA(A) receptor alpha subunits, reduction of GABA(A) receptor binding, and benzodiazepine-resistant anxiety.

Anxiety is a common psychiatric illness often treated by benzodiazepines (BZs). BZs, such as Valium, bind to the alpha subunit of the pentameric GABA(A) receptor and increase inhibition in the CNS. There is considerable evidence for abnormal GABA(A) receptor function in anxiety, and a significant proportion of anxiety patients has a reduced sensitivity to BZs. Here, we show that serotonin(1A) (5-HT(1A)) receptor knock-out mice display BZ-resistant anxiety. Consistent with this finding, binding of both BZ and non-BZ GABA(A) receptor ligands were reduced and GABAergic inhibition was impaired in mutant mice. These changes were reflected by abnormal alpha subunit expression in the amygdala and hippocampus, two important limbic regions involved in fear and anxiety. These data suggest a pathological pathway, initiated by a 5-HT(1A) receptor deficit, leading to abnormalities in GABA(A) receptor composition and level, which in turn result in BZ-insensitivity and anxiety. This model mechanistically links together the 5-HT and GABA systems, which both have been implicated in anxiety. A related mechanism may underlie reduced BZ sensitivity in certain forms of anxiety.

Identification and immunohistochemical mapping of GABAA receptor subtypes containing the delta-subunit in rat brain.

Synaptic inhibition in brain is mainly mediated via GABAA receptors which display a striking structural heterogeneity. A novel type of GABAA receptor subunit, the delta-subunit, has recently been described based on molecular cloning of its cDNA. To identify the prevalence and distribution of GABAA receptors which contain the delta-subunit protein in situ, polyclonal site-directed antisera were developed against three synthetic peptides derived form the rat delta-subunit cDNA-sequence. All antisera specifically recognized a 54 kDa protein in GABAA receptor preparations. Nearly 30% of the GABAA receptors contained the delta-subunit immunoreactivity and displayed high affinity GABA and high affinity benzodiazepine binding sites as shown by immunoprecipitation. Receptors which contain the delta-subunit were immunohistochemically shown to be restricted to a few brain areas such as the cerebellum, thalamus and dentate gyrus of the hippocampal formation. Thus, those neurons which express GABAA receptors with a delta-subunit have now been visualized and made accessible for a functional analysis of this GABAA receptor subtype in situ.

Synapse-specific localization of NMDA and GABA(A) receptor subunits revealed by antigen-retrieval immunohistochemistry.

Conventional immunohistochemistry provides little evidence for the synaptic localization of ionotropic neurotransmitter receptors, suggesting that their epitopes are not readily accessible in situ. Here, we have adapted antigen retrieval procedures based on microwave irradiation to enhance the immunohistochemical staining of gamma-aminobutyric acid type A (GABA[A]) and N-methyl-D-aspartate (NMDA) receptor subunits in rat brain tissue. Microwave irradiation of fixed tissue produced a marked reduction of nonspecific staining, allowing an improved detection of GABA(A) receptor subunits. However, staining of NMDA receptor subunits remained suboptimal. In contrast, microwave irradiation of cryostat sections prepared from fresh tissue resulted in a major enhancement of both NMDA and GABAA receptor subunit staining. The diffuse, partially intracellular signals were largely replaced by numerous, intensely immunoreactive puncta outlining neuronal somata and dendrites, highly suggestive ofsynaptic receptors. In hippocampus CA1-CA3 fields, the NR2Aand NR2B subunit positive puncta exhibited an extensive colocalization in the stratum oriens and radiatum, whereas pyramidal cell bodies, which receive no excitatory synapses, were unstained. In addition, the NR2A subunit, but not the NR2B subunit, was selectively detected on pyramidal cell dendrites in the stratum lucidum of CA3, suggesting a selective targeting to sites of mossy fiber input. For the GABAA receptor subunits, the most striking change induced by this protocol was the selective staining of the axon initial segment of cortical and hippocampal pyramidal cells. The alpha2 subunit immunoreactivity was particularly prominent in these synapses. In control experiments, the staining of cytoskeletal proteins (neurofilaments, glial fibrillary acid protein) was not influenced by prior microwave irradiation. The enhancement of cell-surface-associated staining is therefore strongly suggestive of an 'unmasking' of subunit epitopes by the microwave treatment. These results reveal a remarkable specificity in the synaptic targeting of NMDA and GABAA receptor subunits in hippocampal and neocortical neurons, suggesting that individual neurons can express multiple receptor subtypes in functionally distinct synapses.

GABAA receptor subtypes differentiated by their gamma-subunit variants: prevalence, pharmacology and subunit architecture.

Native GABAA receptors containing different gamma-subunit variants were distinguished immunobiochemically with antisera selectively recognizing the gamma 1-, gamma 2- and gamma 3-subunits. While GABAA receptors containing the gamma 2-subunits were confirmed to be rather ubiquitous in the adult brain, receptors characterized by the gamma 1- or gamma 3-subunit were of low abundance, as shown by immunoprecipitation. The three receptor populations differed strikingly in their benzodiazepine (BZ) site ligand binding profiles. The gamma 3-receptor population displayed reduced affinity for the full agonists clonazepam flunitrazepam and virtually lacked sensitivity to zolpidem. The gamma 1-receptor population displayed low affinity for all benzodiazepine site ligands tested, except for flunitrazepam, and could be differentiated from the gamma 2- and gamma 3-receptors by its low affinity for the inverse agonist beta CCM and its lack of affinity for the partial inverse agonist Ro 15-4513 and the antagonist flumazenil. Since flumazenil antagonizes all major effects of BZ agonists, gamma 1-receptors are not involved in mediating these actions in vivo. In immunopurified receptors, the gamma-subunit variants were found to be assembled with different variants of alpha- and beta-subunits, indicating that not only the gamma 2-subunit gamma 1- and gamma 3-subunits are part of various receptor subtypes. In addition, the gamma 2- and gamma 3-subunits can be co-assembled in native receptors, consistent with the subunit stoichiometry of two alpha-, one beta- and two gamma-subunits proposed previously for recombinant receptors.

[3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors.

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [3H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [3H]CGP 61594 labeled with high affinity (K(D) = 23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [3H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [3H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [3H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors.

The gamma 2 subunit of the GABAA receptor is concentrated in synaptic junctions containing the alpha 1 and beta 2/3 subunits in hippocampus, cerebellum and globus pallidus.

The gamma 2 subunit is necessary for the expression of the full benzodiazepine pharmacology of GABAA receptors and is one of the major subunits in the brain. In order to determine the location of channels containing the gamma 2 subunit in relation to GABA-releasing terminals on the surface of neurons, a new polyclonal antipeptide antiserum was developed to the gamma 2 subunit and used in high resolution, postembedding, immunoelectron-microscopic procedures. Dual immunogold labelling of the same section for two subunits, and up to three sections of the same synapse reacted for different subunits, were used to characterize the subunit composition of synaptic receptors. The gamma 2 subunit was present in type 2, "symmetrical" synapses in each of the brain areas studied, with the exception of the granule cell layer of the cerebellum. The gamma 2 subunit was frequently co-localized in the same synaptic junction with the alpha 1 and beta 2/3 subunits. The immunolabelling of synapses was coincident with the junctional membrane specialization of the active zone. Immunolabelling for the receptor often occurred in multiple clusters in the synapses. In the hippocampus, the gamma 2 subunit was present in basket cell synapses on the somata and proximal dendrites and in axo-axonic cell synapses on the axon initial segment of pyramidal and granule cells. Some synapses on the dendrites of GABAergic interneurones were densely labelled for the gamma 2, alpha 1 and beta 2/3 subunits. In the cerebellum, the gamma 2 subunit was present in both distal and proximal Purkinje cell dendritic synapses established by stellate and basket cell, respectively. On the soma of Purkinje cells, basket cell synapses were only weakly labelled. Synapses on interneuron dendrites were more densely labelled for the gamma 2, alpha 1 and beta 2/3 subunits than synapses on Purkinje or granule cells. Although immunoperoxidase and immunofluorescence methods show an abundance of the gamma 2 subunit in granule cells, the labelling of Golgi synapses was much weaker with the immunogold method than that of the other cell types. In the globus pallidus, many type 2 synapses were labelled for the gamma 2 subunit together with alpha 1 and beta 2/3 subunits. The results show that gamma 2 and beta 2/3 subunits receptor channels are highly concentrated in GABAergic synapses that also contain the alpha 1 and beta 2/3 subunits. Channels containing the gamma 2 subunit are expressed in synapses on functionally distinct domains of the same neuron receiving GABA from different presynaptic sources. There are quantitative differences in the density of GABAA receptors at synapses on different cell types in the same brain area.

Neuron-specific expression of GABAA-receptor subtypes: differential association of the alpha 1- and alpha 3-subunits with serotonergic and GABAergic neurons.

GABAA-receptors in the brain display a striking structural heterogeneity, which is based on a multiplicity of diverse subunits. The allocation of GABAA-receptor subtypes to identified neurons is essential for an analysis of the functional significance of receptor heterogeneity. Among GABA-receptive neurons, well-characterized examples include the serotonergic and GABAergic neurons in the raphe nuclei. The GABAA-receptor subtypes expressed in these two types of neurons were analysed using antisera which recognize selectively the alpha 1- and alpha 3-subunits, and their co-localization with serotonin and glutamate decarboxylase was assessed by confocal laser microscopy in double and triple immunofluorescence staining in the rat. The vast majority of serotonergic neurons express strong alpha 3-subunit-immunoreactivity, but are devoid of alpha 1-subunit staining. In contrast, both the alpha 1- and alpha 3-subunit-immunoreactivities are present in glutamate decarboxylase-positive neurons. Thus, serotonergic and GABAergic neurons selectively express distinct patterns of alpha subunits, suggesting that they possess distinct subtypes of GABAA-receptors. The occurrence of neuron-specific GABAA-receptor subtypes may open new possibilities for the targeting of drugs with selective therapeutic actions.

N-methyl-D-aspartate receptors containing the NR2D subunit in the retina are selectively expressed in rod bipolar cells.

N-methyl-D-aspartate receptor subunit messenger RNAs are widely expressed in the retina and several types of second and third order neurons are responsive to N-methyl-D-aspartate. Functional N-methyl-D-aspartate receptors are assembled from the NR1 subunit with at least one of the four NR2 subunit variants (NR2A-2D). We have analysed immunohistochemically the cellular distribution of N-methyl-D-aspartate receptors containing the NR2D subunit in the rat and rabbit retina. Using a subunit-specific NR2D antiserum, exclusively bipolar cells with somata localized close to the outer plexiform layer were labelled in both species. The axons were immunoreactive and arborized in the innermost inner plexiform layer. The morphology and localization of these cells, which were much more numerous in rat than in rabbit, suggested that they are rod bipolar cells. This was confirmed in both species by co-localization of the NR2D subunit immunoreactivity with protein kinase C-alpha, a selective marker for rod bipolar cells. At the subcellular level, a distinct polarization in the distribution of NR2D immunoreactivity was demonstrated by confocal laser scanning microscopy: staining was moderate in dendrites arborizing within the outer plexiform layer, intense at that pole of the soma facing the outer plexiform layer, and low in the portion of the soma embedded in the inner nuclear layer. Proximal axonal segments and axonal end-feet in the inner plexiform layer displayed the strongest NR2D subunit immunoreactivity. The axonal staining suggests that neurotransmission of the rod bipolar cells is modulated within the inner plexiform layer by N-methyl-D-aspartate receptors containing the NR2D subunit.

GABAA-receptor alpha-subunit is an essential prerequisite for receptor formation in vivo.

The mechanisms governing the assembly of alpha-, beta- and gamma-subunits to form GABAA-receptors are poorly understood. Here, we report that the alpha-subunit is essential for receptor assembly. In mice homozygous for a deletion on chromosome 7 spanning the alpha 5- and gamma 3-subunit genes, zolpidem-insensitive benzodiazepine binding sites, corresponding to GABAA-receptors containing the alpha 5-subunit, were absent in the hippocampus. This loss of alpha 5-GABAA-receptor binding was also apparent as a 21% decrease in the total number of benzodiazepine binding sites in the hippocampus. In addition, immunoreactivity for the beta 2,3- and gamma 2-subunit was decreased exclusively in neurons which normally express the alpha 5-subunit, such as olfactory bulb granule cells and hippocampal pyramidal cells. In other brain regions of the mutants, the beta 2,3- and gamma 2-subunit staining was unaffected. Controls included two lines of mice homozygous for a shorter chromosomal deletion, that either included or excluded the gamma 3-subunit gene. These two lines were indistinguishable with regard to numbers of benzodiazepine binding sites and levels alpha 5-, beta 2,3- and gamma 2-subunit immunoreactivity, indicating that the lack of gamma 3-subunit gene did not contribute to the observed deficit in receptor formation. These results demonstrate that the absence of the alpha 5-subunit gene prevents the formation of the entire respective receptor complex in adult mouse brain. Thus, the alpha-subunit, unlike the gamma 2-subunit, might play a major role in the assembly or targeting of GABAA-receptor complexes.

Protein-chemical analysis of Bio-Oss bone substitute and evidence on its carbonate content.

The natural bone substitute Bio-Oss is used in periodontal and maxillofacial surgery to fill bone defects and permit reossification. Recent reports have suggested the presence of TGFbeta and of substantial amounts of protein in Bio-Oss and have questioned its position as a biologically inert material and its safety in clinical applications (Hönig et al., Plast Reconstr Surg 1999;103:1324; Schwartz et al., J Periodontol 2000;71:1258). Bio-Oss was therefore subjected to a detailed biochemical, histochemical and biophysical analysis. In three different types of extracts of Bio-Oss no evidence for the presence of protein based on SDS-PAGE and silver staining was detected. In addition, as shown by Western blotting, there was no immunochemical evidence for the presence of the potential growth-inducing factor TGFbeta. Furthermore, micropolished sections of Bio-Oss failed to be stained with McNeal's Tetrachrome as did microtome sections treated with Goldner's Trichrome. However, Bio-Oss was strongly stained with the protein dye Coomassie blue. This staining was virtually irreversible and is attributed to the carbonate content of Bio-Oss which was detected by thermogravimetry-mass spectrometry. Thus, within the limits of the assay conditions, Bio-Oss does not contain protein material to a measurable extent.

Identification of an endogenous polypeptide modulating ligand binding sites of insect neuronal acetylcholine receptors.

In this report evidence is presented for a membrane-associated polypeptide that regulates ligand binding properties of a neuronal acetylcholine receptor. Removal of membrane-associated compounds reversibly increased the number of BGTX binding sites and decreased the number of binding sites for ACh in neuronal membranes, suggesting the existence of endogenous membrane-associated factors that might allosterically modulate the ligand binding sites of receptor protein. The regulatory factor was purified and identified as 20 kDa polypeptide. The purified polypeptide was found to be phosphorylated by cAMP-dependent protein kinase, which caused inactivation of the modulatory polypeptide and thus might control their function.

Distribution of NMDA receptor subunit proteins NR2A, 2B, 2C and 2D in rat brain.

The regional distribution of the NMDA receptor subunits NR2A, 2B, 2C and 2D was visualized in adult rat brain using the histo-blot technique with newly developed subunit-specific antisera. NR2A immunoreactivity was found in almost all regions of the brain, whereas NR2B staining was restricted to forebrain, and NR2D immunoreactivity to diencephalic, mesencephalic and brain stem structures. NR2C staining was confined to cerebellum, thalamus and olfactory bulb. Thus, NMDA receptors containing the NR2A subunit are likely to represent a receptor subtype predominant throughout the brain, while those containing the NR2B, NR2C or NR2D subunit represent more region-specific receptor subtypes. The regionally overlapping distribution of certain NR2 subunits points to the existence of NMDA receptors containing more than one NR2 subunit variant.

Messenger RNA from insect nervous tissue induces expression of neuronal acetylcholine receptors in Xenopus oocytes.

mRNA isolated from the nervous tissue of young insects microinjected in Xenopus oocytes induced the expression of alpha-toxin binding sites which were inserted into the surface membrane. Immunoprecipitation experiments revealed that the coded receptor polypeptides were processed to the authentic size, and ion flux studies demonstrated that functional nicotinic acetylcholine receptors were produced and inserted into the oocyte membrane.

Identification of the gamma 2-subunit protein in native GABAA receptors in brain.

GABAA receptors with functional benzodiazepine receptors have been reconstituted by coexpression of alpha-, beta- and gamma-subunit cDNAs. In brain, proteins of the alpha- and beta-subunits, but not the gamma 2-subunit have been identified. Using an antipeptide antiserum, we now demonstrate that the gamma 2-subunit is a protein of 43 kDa. It is present in at least 50% of GABAA receptors, as shown by immunoprecipitation.

Photoaffinity labeling of the NMDA receptor.

The structure of NMDA receptors in situ has been probed with the novel photoaffinity ligand 125I-CGP 55802A. By covalently linking the radioactive high-affinity photolabel to NMDA receptors in bovine brain we have identified a protein of 175 kDa associated with the binding site for NMDA receptor agonists and competitive antagonists. Based on its molecular size the photolabeled protein is likely to correspond to the NR2A and/or NR2B subunit. The photoaffinity ligand will permit the assessment of regulatory changes in NMDA receptor subunit expression.