Homeostasis describes the fundamental biological ability of individuals to maintain stable internal conditions in a changing environment. Homeostatic reactions include internal adjustments as well as behavioral responses. In vertebrates, behavioral responses are induced by the reward system. This system originates in the ventral tegmental area (VTA) and leads to increased dopamine levels in the forebrain whenever activated. A major inhibitor of VTA activity is the lateral habenula (LHb). This epithalamic structure is able to almost completely suppress dopamine release, either directly or via the rostromedial tegmental nucleus (RMTg), when rewarding expectations are not met. A major input to the LHb arises from the lateral hypothalamic area (LHA), an important regulator of the homeostatic system. Currently, little is known about the effects of the strong hypothalamic projection on the activity of LHb neurons. In the present study, we analyze neurotransmitters and cellular targets of the LHA-LHb projection in the rat. Therefore, anterograde tracing from the LHA was combined with the visualization of neurotransmitters in the LHb. These experiments revealed a mainly glutamatergic projection, probably exerting excitatory effects on the targeted LHb cells. These cellular targets were analyzed in a second step. Anterograde tracing from the LHA in combination with retrograde tracing from the VTA/RMTg region revealed that LHb neurons projecting to the VTA/RMTg region are densely targeted by the LHA projection. Visualization of synaptophysin at these contact sites indicates that the contact sites indeed are synapses. Taken together, the present study describes a strong mainly glutamatergic projection from the LHA that targets VTA/RMTg-projecting neurons in the LHb. These findings emphasize the potential role of the LHb as direct link between homeostatic areas and reward circuitries, which may be important for the control of homeostatic behaviors.

TipE is an auxiliary subunit of the Drosophila Para sodium channel. Here we describe four sequences, TEH1-4, homologous to TipE in the Drosophila melanogaster genome, harboring all typical structures of both TipE and the beta-Subunit family of big-conductance Ca(2+)-activated potassium channels: short cytosolic N- and C-terminal stretches, two transmembrane domains, and a large extracellular loop with two disulfide bonds. Whereas TEH1 and TEH2 lack the TipE-specific extension in the extracellular loop, both TEH3 and TEH4 possess two extracellular EGF-like domains. A CNS-specific expression was found for TEH1, while TEH2-4 were more widely expressed. The genes for TEH2-4 are localized close to the tipE gene on chromosome 3L. Coexpression of TEH subunits with Para in Xenopus oocytes showed a strong (30-fold, TEH1), medium (5- to 10-fold, TEH2 and TEH3), or no (TEH4) increase in sodium current amplitude, while TipE increased the current 20-fold. In addition, steady-state inactivation and the recovery from fast inactivation were altered by coexpression of Para with TEH1. We conclude that members of the TEH-family are auxiliary subunits for Para sodium channels and possibly other ion channels.

Interaction with biological material can alter physicochemical parameters of magnetic nanoparticles and might thereby change their magnetic behavior with potentially important implications for various nanoparticle applications. Little is known about changes of the magnetic behavior that occur during the initial phase of cell binding and uptake. We investigate the magnetic behavior of very small superparamagnetic iron-oxide nanoparticles (VSOP) during initial contact with THP-1 monocytes. We combine real-time magnetic particle spectroscopy (MPS), a fast and sensitive method for specific detection of magnetic nanoparticles in biological specimen with high-pressure-freezing/freeze-substitution transmission electron microscopy (HPF/FS-TEM), enabling us to generate snapshots of the interaction of VSOP with the cellular glycocalyx. MPS reveals significant changes of the dynamic magnetic behavior within seconds after VSOP injection into monocyte suspensions that correlate with the formation of nanoparticle clusters in the glycocalyx. The combination of real-time MPS and HPF/FS-TEM provides an ideal platform to analyze magnetic behaviors of nanoparticles upon interaction with cells and tissues.

Bilaterians usually possess a central nervous system, composed of neurons and supportive cells called glial cells. Whereas neuronal cells are highly comparable in all these animals, glial cells apparently differ, and in deuterostomes, radial glial cells are found. These particular secretory glial cells may represent the archetype of all (macro) glial cells and have not been reported from protostomes so far. This has caused controversial discussions of whether glial cells represent a homologous bilaterian characteristic or whether they (and thus, centralized nervous systems) evolved convergently in the two main clades of bilaterians. By using histology, transmission electron microscopy, immunolabelling and whole-mount in situ hybridization, we show here that protostomes also possess radial glia-like cells, which are very likely to be homologous to those of deuterostomes. Moreover, our antibody staining indicates that the secretory character of radial glial cells is maintained throughout their various evolutionary adaptations. This implies an early evolution of radial glial cells in the last common ancestor of Protostomia and Deuterostomia. Furthermore, it suggests that an intraepidermal nervous system-composed of sensory cells, neurons and radial glial cells-was probably the plesiomorphic condition in the bilaterian ancestor.

The interest in astrocytes, the silent brain cells that accumulate polyamines (PAs), is growing. PAs exert anti-inflammatory, antioxidant, antidepressant, neuroprotective, and other beneficial effects, including increasing longevity in vivo. Unlike neurons, astrocytes are extensively coupled to others via connexin (Cx) gap junctions (GJs). Although there are striking modulatory effects of PAs on neuronal receptors and channels, PA regulation of the astrocytic GJs is not well understood. We studied GJ-propagation using molecules of different (i) electrical charge, (ii) structure, and (iii) molecular weight. Loading single astrocytes with patch pipettes containing membrane-impermeable dyes, we observed that (i) even small molecules do not easily permeate astrocytic GJs, (ii) the ratio of the charge to weight of these molecules is the key determinant of GJ permeation, (iii) the PA spermine (SPM) induced the propagation of negatively charged molecules via GJs, (iv) while no effects were observed on propagation of macromolecules with net-zero charge. The GJ uncoupler carbenoxolone (CBX) blocked such propagation. Taken together, these findings indicate that SPM is essential for astrocytic GJ communication and selectively facilitates intracellular propagation via GJs for negatively charged molecules through glial syncytium.

Optimization of the AV-interval (AVI) in DDD pacemakers improves cardiac hemodynamics and reduces pacemaker syndromes. Manual optimization is typically not performed in clinical routine. In the present study we analyze the prevalence of E/A wave fusion and A wave truncation under resting conditions in 160 patients with complete AV block (AVB) under the pre-programmed AVI. We manually optimized sub-optimal AVI.

Retinal ON bipolar cells make up about 70% of all bipolar cells. Glutamate hyperpolarizes these cells by binding to the metabotropic glutamate receptor mGluR6, activating the G-protein G(o1), and closing an unidentified cation channel. To facilitate investigation of ON bipolar cells, we here report on the production of a transgenic mouse (Grm6-GFP) in which enhanced green fluorescent protein (EGFP), under control of mGluR6 promoter, was expressed in all and only ON bipolar cells. We used the mouse to determine density of ON bipolar cells, which in central retina was 29,600 cells/mm(2). We further sorted the fluorescent cells and created a pure ON bipolar cDNA library that was negative for photoreceptor unique genes. With this library, we determined expression of 27 genes of interest. We obtained positive transcripts for G(o) interactors: regulators of G-protein signaling (RGS), Ret-RGS1 (a variant of RGS20), RGS16, RGS7, purkinje cell protein 2 (PCP2, also called L7 or GPSM4), synembryn (RIC-8), LGN (GPSM2), RAP1GAP, and Gbeta5; cGMP modulators: guanylyl cyclase (GC) 1alpha1, GC1beta1, phosphodiesterase (PDE) 1C, and PDE9A; and channels: inwardly rectifying potassium channel Kir2.4, transient receptor potential TRPC2, and sperm-specific cation channels CatSper 2-4. The following transcripts were not found in our library: AGS3 (GPSM1), RGS10, RGS19 (GAIP), calbindin, GC1alpha2, GC1beta2, PDE5, PDE2A, amiloride-sensitive sodium channel ACCN4, and CatSper1. We then localized Kir2.4 to several cell types and showed that, in ON bipolar cells, the channel concentrates in their dendritic tips. The channels and modulators found in ON bipolar cells likely shape their light response. Additional uses of the Grm6-GFP mouse are also discussed.

The evolutionary conserved NEAT1-MALAT1 gene cluster generates large noncoding transcripts remaining nuclear, while tRNA-like transcripts (mascRNA, menRNA) enzymatically generated from these precursors translocate to the cytosol. Whereas functions have been assigned to the nuclear transcripts, data on biological functions of the small cytosolic transcripts are sparse. We previously found NEAT1-/- and MALAT1-/- mice to display massive atherosclerosis and vascular inflammation. Here, employing selective targeted disruption of menRNA or mascRNA, we investigate the tRNA-like molecules as critical components of innate immunity. CRISPR-generated human ΔmascRNA and ΔmenRNA monocytes/macrophages display defective innate immune sensing, loss of cytokine control, imbalance of growth/angiogenic factor expression impacting upon angiogenesis, and altered cell-cell interaction systems. Antiviral response, foam cell formation/oxLDL uptake, and M1/M2 polarization are defective in ΔmascRNA/ΔmenRNA macrophages, defining first biological functions of menRNA and describing new functions of mascRNA. menRNA and mascRNA represent novel components of innate immunity arising from the noncoding genome. They appear as prototypes of a new class of noncoding RNAs distinct from others (miRNAs, siRNAs) by biosynthetic pathway and intracellular kinetics. Their NEAT1-MALAT1 region of origin appears as archetype of a functionally highly integrated RNA processing system.

Anti-NMDA receptor encephalitis was first described about thirteen years ago and has become one of the most important differential diagnoses for new-onset psychosis. The disease is mediated by autoantibodies against the subunit 1 of the N-methyl-D-aspartate receptor (NMDA-R1) in patients presenting with variable clinical symptoms. Patients often profit from immunmodulatory therapy, independent of their individual symptoms. In this study CSF samples as well as monoclonal antibodies derived from patients diagnosed with NMDA-R1 encephalitis were applied to rat hippocampus and visualized by immunocytochemistry. This reveals at least two distinct patterns of immunoreactivity. Antibodies from "pattern group 1" display the familiar pattern of NMDA-R1 distribution in the hippocampus reported in experiments with rabbit anti-NMDA-R1 antibodies. Neurons and primary dendrites in the CA1 and CA3 region show strongly stained cell bodies, in line with the predominant postsynaptic localization of the NMDA receptor in the brain. However, autoantibodies from "pattern group 2" show an inverse pattern, with no staining of the cell bodies and primary dendrites in CA1 and CA3 regions. Electron microscopic experiments disclose that autoantibodies of "pattern group 1 patients" bind to postsynaptic NMDA receptors, while those of "pattern group 2 patients" target presynaptic NMDA receptors. We describe one NMDA-receptor antibody giving staining comparable to rabbit anti-NMDA-R1 antibodies, raised against the C-terminus. In the highly heterogenous disease anti-NMDA-receptor 1 encephalitis we found evidence for at least two different subtypes. It will be very interesting to determine whether there also are two distinct clinical phenotypes.

The striatum is the main input structure of the basal ganglia. Distinct striatal subfields are involved in voluntary movement generation and cognitive and emotional tasks, but little is known about the morphological and molecular differences of striatal subregions. The ventrolateral subfield of the striatum (VLS) is the orofacial projection field of the sensorimotor cortex and is involved in the development of orofacial dyskinesias, involuntary chewing-like movements that often accompany long-term neuroleptic treatment. The biological basis for this particular vulnerability of the VLS is not known. Potassium channels are known to be strategically localized within the striatum. In search of possible molecular correlates of the specific vulnerability of the VLS, we analyzed the expression of voltage-gated potassium channels in rodent and primate brains using qPCR, in situ hybridization, and immunocytochemical single and double staining. Here we describe a novel, giant, non-cholinergic interneuron within the VLS. This neuron coexpresses the vesicular GABA transporter, the calcium-binding protein parvalbumin (PV), and the Kv3.3 potassium channel subunit. This novel neuron is much larger than PV neurons in other striatal regions, displays characteristic electrophysiological properties, and, most importantly, is restricted to the VLS. Consequently, the giant striatal Kv3.3-expressing PV neuron may link compromised Kv3 channel function and VLS-based orofacial dyskinesias.

Communication within the glial cell ecosystem is essential for neuronal and brain health1-3. The influence of glial cells on the accumulation and clearance of β-amyloid (Aβ) and neurofibrillary tau in the brains of individuals with Alzheimer's disease (AD) is poorly understood, despite growing awareness that these are therapeutically important interactions4,5. Here we show, in humans and mice, that astrocyte-sourced interleukin-3 (IL-3) programs microglia to ameliorate the pathology of AD. Upon recognition of Aβ deposits, microglia increase their expression of IL-3Rα-the specific receptor for IL-3 (also known as CD123)-making them responsive to IL-3. Astrocytes constitutively produce IL-3, which elicits transcriptional, morphological, and functional programming of microglia to endow them with an acute immune response program, enhanced motility, and the capacity to cluster and clear aggregates of Aβ and tau. These changes restrict AD pathology and cognitive decline. Our findings identify IL-3 as a key mediator of astrocyte-microglia cross-talk and a node for therapeutic intervention in AD.

Hyperexcitability in the orbitofrontal cortex (OFC) is a key clinical feature of anhedonic domains of Major Depressive Disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unknown. Here, cell-population-specific chromatin accessibility profiling in human OFC unexpectedly mapped genetic risk for MDD exclusively to non-neuronal cells, and transcriptomic analyses revealed significant glial dysregulation in this region. Characterization of MDD-specific cis-regulatory elements identified ZBTB7A - a transcriptional regulator of astrocyte reactivity - as an important mediator of MDD-specific chromatin accessibility and gene expression. Genetic manipulations in mouse OFC demonstrated that astrocytic Zbtb7a is both necessary and sufficient to promote behavioral deficits, cell-type-specific transcriptional and chromatin profiles, and OFC neuronal hyperexcitability induced by chronic stress - a major risk factor for MDD. These data thus highlight a critical role for OFC astrocytes in stress vulnerability and pinpoint ZBTB7A as a key dysregulated factor in MDD that mediates maladaptive astrocytic functions driving OFC hyperexcitability.

In humans, temporal lobe epilepsy (TLE) is often associated with Ammon's horn sclerosis (AHS) characterized by hippocampal cell death, gliosis and granule cell dispersion (GCD) in the dentate gyrus. Granule cells surviving TLE have been proposed to be hyperexcitable and to play an important role in seizure generation. However, it is unclear whether this applies to conditions of AHS. We studied granule cells using the intrahippocampal kainate injection mouse model of TLE, brain slice patch-clamp recordings, morphological reconstructions and immunocytochemistry. With progressing AHS and GCD, 'epileptic' granule cells of the injected hippocampus displayed a decreased input resistance, a decreased membrane time constant and an increased rheobase. The resting leak conductance was doubled in epileptic granule cells and roughly 70-80% of this difference were sensitive to K(+) replacement. Of the increased K(+) leak, about 50% were sensitive to 1 mm Ba(2+). Approximately 20-30% of the pathological leak was mediated by a bicuculline-sensitive GABA(A) conductance. Epileptic granule cells had strongly enlarged inwardly rectifying currents with a low micromolar Ba(2+) IC(50), reminiscent of classic inward rectifier K(+) channels (Irk/Kir2). Indeed, protein expression of Kir2 subunits (Kir2.1, Kir2.2, Kir2.3, Kir2.4) was upregulated in epileptic granule cells. Immunolabelling for two-pore weak inward rectifier K(+) channels (Twik1/K2P1.1, Twik2/K2P6.1) was also increased. We conclude that the excitability of granule cells in the sclerotic focus of TLE is reduced due to an increased resting conductance mainly due to upregulated K(+) channel expression. These results point to a local adaptive mechanism that could counterbalance hyperexcitability in epilepsy.

Osmotic swelling of retinal glial (Müller) cells may contribute to the development of edema in diabetic retinopathy. Here, we tested whether oxidative stress and mitochondrial dysfunction are pathogenic factors involved in the osmotic swelling of Müller cells in retinal slices from control and streptozotocin-injected hyperglycemic rats. Hypotonic challenge did not change the size of Müller cell somata from control animals but induced soma swelling in Müller cells of diabetic animals. Administration of a reducing agent blocked the osmotic swelling of Müller cell somata. In retinal tissues from control animals, administration of the reducing agent blocked also the swelling-inducing effects of antagonists of P2Y₁ and adenosine A₁ receptors. In tissues from diabetic animals, inhibition of xanthine oxidase decreased the soma swelling by approximately 50% while inhibition of NADPH oxidase and nitric oxide synthase had no effects. Blockade of mitochondrial oxidative stress by perindopril, as well as of mitochondrial permeability transition by cyclosporin A or minocycline, attenuated the swelling. In addition, activation of mitochondrial K(ATP) channels by pinacidil fully prevented the swelling. The data suggest that oxidative stress produced by xanthine oxidase, as well as the mitochondria, are implicated in the induction of osmotic swelling of Müller cells from diabetic rats.

The lateral habenular complex (LHb) is a bilateral epithalamic brain structure involved in the modulation of ascending monoamine systems in response to afferents from limbic regions and basal ganglia. The LHb is implicated in various biological functions, such as reward, sleep-wake cycle, feeding, pain processing, and memory formation. The modulatory role of the LHb is partially assumed by putative spontaneously active LHb neurons projecting to the dopaminergic ventral tegmental area (VTA) and to the serotonergic median (MnR) and dorsal raphe nuclei (DR). All four nuclei form a complex and coordinated network to evoke appropriate responses to reward-related stimuli. At present it is not known whether individual LHb neurons project to only one or to more than one monoaminergic nucleus. To answer this question, we made dual injections of two different retrograde tracers into the rat VTA and either DR or MnR. Tracers were visualized by immunohistochemistry. In coronal sections, the different retrogradly labeled habenular neurons were quantified and assigned to the corresponding habenular subnuclei. Our results show that 1) the distribution of neurons in the LHb projecting to the three monoamine nuclei is similar and exhibits a great overlap, 2) the vast majority of LHb projection neurons target one monoaminergic nucleus only, and 3) very few, heterogeneously distributed LHb neurons project to both dopaminergic and serotonergic nuclei. These results imply that the LHb forms both separate and interconnected circuits with each monoaminergic nucleus, permitting the LHb to modulate its output to different monoamine systems either independently or jointly.

Optical coherence tomography (OCT) is an intravascular, high-resolution imaging technique that is used to characterize atherosclerotic plaques. However, the identification of macrophages as important markers of inflammation and plaque vulnerability remains difficult. Here, we investigate whether the uptake of very small iron oxide particles (VSOP) in macrophages, that cluster in phagolysosomes and allow high-quality magnetic resonance imaging (MRI) of atherosclerotic plaques, and uptake of ferumoxytol nanoparticles enhance detection of macrophages by OCT.

The dorsal diencephalic conduction system connects limbic forebrain structures to monaminergic mesencephalic nuclei via a distinct relay station, the habenular complexes. Both habenular nuclei, the lateral as well as the medial nucleus, are considered to play a prominent role in mental disorders like major depression. Herein, we investigate the effect of the polyamine agmatine on the electrical activity of neurons within the medial habenula in rat. We present evidence that agmatine strongly decreases spontaneous action potential firing of medial habenular neurons by activating I1-type imidazoline receptors. Additionally, we compare the expression patterns of agmatinase, an enzyme capable of inactivating agmatine, in rat and human habenula. In the medial habenula of both species, agmatinase is similarly distributed and observed in neurons and, in particular, in distinct neuropil areas. The putative relevance of these findings in the context of depression is discussed. It is concluded that increased activity of the agmatinergic system in the medial habenula may strengthen midbrain dopaminergic activity. Consequently, the habenular-interpeduncular axis may be dysregulated in patients with major depression.

The biochemical response to food intake must be precisely regulated. Because ingested sugars and fats can feed into many anabolic and catabolic pathways1, how our bodies handle nutrients depends on strategically positioned metabolic sensors that link the intrinsic nutritional value of a meal with intermediary metabolism. Here we describe a subset of immune cells-integrin β7+ natural gut intraepithelial T lymphocytes (natural IELs)-that is dispersed throughout the enterocyte layer of the small intestine and that modulates systemic metabolism. Integrin β7- mice that lack natural IELs are metabolically hyperactive and, when fed a high-fat and high-sugar diet, are resistant to obesity, hypercholesterolaemia, hypertension, diabetes and atherosclerosis. Furthermore, we show that protection from cardiovascular disease in the absence of natural IELs depends on the enteroendocrine-derived incretin GLP-12, which is normally controlled by IELs through expression of the GLP-1 receptor. In this metabolic control system, IELs modulate enteroendocrine activity by acting as gatekeepers that limit the bioavailability of GLP-1. Although the function of IELs may prove advantageous when food is scarce, present-day overabundance of diets high in fat and sugar renders this metabolic checkpoint detrimental to health.

The thymus is a primary lymphoid organ necessary for optimal T cell development. Here, we show that liver X receptors (LXRs)-a class of nuclear receptors and transcription factors with diverse functions in metabolism and immunity-critically contribute to thymic integrity and function. LXRαβ-deficient mice develop a fatty, rapidly involuting thymus and acquire a shrunken and prematurely immunoinhibitory peripheral T cell repertoire. LXRαβ's functions are cell specific, and the resulting phenotypes are mutually independent. Although thymic macrophages require LXRαβ for cholesterol efflux, thymic epithelial cells (TECs) use LXRαβ for self-renewal and thymocytes for negative selection. Consequently, TEC-derived LXRαβ protects against homeostatic premature involution and orchestrates thymic regeneration following stress, while thymocyte-derived LXRαβ limits cell disposal during negative selection and confers heightened sensitivity to experimental autoimmune encephalomyelitis. These results identify three distinct but complementary mechanisms by which LXRαβ governs T lymphocyte education and illuminate LXRαβ's indispensable roles in adaptive immunity.

Polyamines (PAs) are polycationic biomolecules containing multiple amino groups. Patients with HIV-associated neurocognitive disorder (HAND) have high concentrations of the polyamine N-acetylated spermine in their brain and cerebral spinal fluid (CSF) and have increased PA release from astrocytes. These effects are due to the exposure to HIV-Tat. In healthy adult brain, PAs are accumulated but not synthesized in astrocytes, suggesting that PAs must enter astrocytes to be N-acetylated and released. Therefore, we tested if Cx43 hemichannels (Cx43-HCs) are pathways for PA flux in control and HIV-Tat-treated astrocytes. We used biotinylated spermine (b-SPM) to examine polyamine uptake. We found that control astrocytes and those treated with siRNA-Cx43 took up b-SPM, similarly suggesting that PA uptake is via a transporter/channel other than Cx43-HCs. Surprisingly, astrocytes pretreated with both HIV-Tat and siRNA-Cx43 showed increased accumulation of b-SPM. Using a novel polyamine transport inhibitor (PTI), trimer 44NMe, we blocked b-SPM uptake, showing that PA uptake is via a PTI-sensitive transport mechanism such as organic cation transporter. Our data suggest that Cx43 HCs are not a major pathway for b-SPM uptake in the condition of normal extracellular calcium concentration but may be involved in the release of PAs to the extracellular space during viral infection.