Thomas Wultsch

STIM2 regulates capacitive Ca2+ entry in neurons and plays a key role in hypoxic neuronal cell death.

Neurons lacking the calcium sensor STIM2 are protected from hypoxia-induced cell death. Resisting Ischemia Loss of blood flow to the brain—as can occur during a stroke—leads to the death of neurons, a process that involves a pathological increase in intracellular calcium. Berna-Erro et al. investigated the role of capacitive calcium entry (CCE), a process in which depletion of calcium from intracellular stores triggers its entry across the plasma membrane, in ischemia-induced calcium entry and neuronal death. The calcium-sensing molecule STIM1 is known to play a crucial role in mediating CCE in various cell types; in neurons, however, Berna-Erro et al. found that CCE depended instead on the closely related molecule STIM2. Neurons from mice lacking STIM2 were resistant to the effects of hypoxia in vitro; moreover, mice lacking STIM2 showed less neurological damage than did wild-type mice in a model of ischemic stroke. Thus, the authors conclude that STIM2 is critical to neuronal CCE and that CCE plays a role in neuronal death in ischemia. Excessive cytosolic calcium ion (Ca2+) accumulation during cerebral ischemia triggers neuronal cell death, but the underlying mechanisms are poorly understood. Capacitive Ca2+ entry (CCE) is a process whereby depletion of intracellular Ca2+ stores causes the activation of plasma membrane Ca2+ channels. In nonexcitable cells, CCE is controlled by the endoplasmic reticulum (ER)–resident Ca2+ sensor STIM1, whereas the closely related protein STIM2 has been proposed to regulate basal cytosolic and ER Ca2+ concentrations and make only a minor contribution to CCE. Here, we show that STIM2, but not STIM1, is essential for CCE and ischemia-induced cytosolic Ca2+ accumulation in neurons. Neurons from Stim2−/− mice showed significantly increased survival under hypoxic conditions compared to neurons from wild-type controls both in culture and in acute hippocampal slice preparations. In vivo, Stim2−/− mice were markedly protected from neurological damage in a model of focal cerebral ischemia. These results implicate CCE in ischemic neuronal cell death and establish STIM2 as a critical mediator of this process.

Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition

Synaptic inhibition is a central factor in the fine tuning of neuronal activity in the central nervous system. Symptoms consistent with reduced inhibition such as stiffness, spasms and anxiety occur in paraneoplastic stiff person syndrome with autoantibodies against the intracellular synaptic protein amphiphysin. Here we show that intrathecal application of purified anti-amphiphysin immunoglobulin G antibodies induces stiff person syndrome-like symptoms in rats, including stiffness and muscle spasms. Using in vivo recordings of Hoffmann reflexes and dorsal root potentials, we identified reduced presynaptic GABAergic inhibition as an underlying mechanism. Anti-amphiphysin immunoglobulin G was internalized into neurons by an epitope-specific mechanism and colocalized in vivo with presynaptic vesicular proteins, as shown by stimulation emission depletion microscopy. Neurons from amphiphysin deficient mice that did not internalize the immunoglobulin provided additional evidence of the specificity in antibody uptake. GABAergic synapses appeared more vulnerable than glutamatergic synapses to defective endocytosis induced by anti-amphiphysin immunoglobulin G, as shown by increased clustering of the endocytic protein AP180 and by defective loading of FM 1-43, a styryl dye used to label cell membranes. Incubation of cultured neurons with anti-amphiphysin immunoglobulin G reduced basal and stimulated release of γ-aminobutyric acid substantially more than that of glutamate. By whole-cell patch-clamp analysis of GABAergic inhibitory transmission in hippocampus granule cells we showed a faster, activity-dependent decrease of the amplitude of evoked inhibitory postsynaptic currents in brain slices treated with antibodies against amphiphysin. We suggest that these findings may explain the pathophysiology of the core signs of stiff person syndrome at the molecular level and show that autoantibodies can alter the function of inhibitory synapses in vivo upon binding to an intraneuronal key protein by disturbing vesicular endocytosis.

Reduced anxiety‐like and depression‐related behavior in neuropeptide Y Y4 receptor knockout mice

Neuropeptide Y (NPY) acting through Y1 receptors reduces anxiety‐ and depression‐like behavior in rodents, whereas Y2 receptor stimulation has the opposite effect. This study addressed the implication of Y4 receptors in emotional behavior by comparing female germ line Y4 knockout (Y4−/−) mice with control and germ line Y2−/− animals. Anxiety‐ and depression‐like behavior was assessed with the open field (OF), elevated plus maze (EPM), stress‐induced hyperthermia (SIH) and tail suspension tests (TST), respectively. Learning and memory were evaluated with the object recognition test (ORT). In the OF and EPM, both Y4−/− and Y2−/− mice exhibited reduced anxiety‐related behavior and enhanced locomotor activity relative to control animals. Locomotor activity in a familiar environment was unchanged in Y4−/− but reduced in Y2−/− mice. The basal rectal temperature exhibited diurnal and genotype‐related alterations. Control mice had temperature minima at noon and midnight, whereas Y4−/− and Y2−/− mice displayed only one temperature minimum at noon. The magnitude of SIH was related to time of the day and genotype in a complex manner. In the TST, the duration of immobility was significantly shorter in Y4−/− and Y2−/− mice than in controls. Object memory 6 h after initial exposure to the ORT was impaired in Y2−/− but not in Y4−/− mice, relative to control mice. These results show that genetic deletion of Y4 receptors, like that of Y2 receptors, reduces anxiety‐like and depression‐related behavior. Unlike Y2 receptor knockout, Y4 receptor knockout does not impair object memory. We propose that Y4 receptors play an important role in the regulation of behavioral homeostasis.

Deletion of the acid-sensing ion channel ASIC3 prevents gastritis-induced acid hyperresponsiveness of the stomach-brainstem axis.

&NA; Gastric acid challenge of the rat and mouse stomach is signalled to the brainstem as revealed by expression of c‐Fos. The molecular sensors relevant to the detection of gastric mucosal acidosis are not known. Since the acid‐sensing ion channels ASIC2 and ASIC3 are expressed by primary afferent neurons, we examined whether knockout of the ASIC2 or ASIC3 gene modifies afferent signalling of a gastric acid insult in the normal and inflamed stomach. The stomach of conscious mice (C57BL/6) was challenged with intragastric HCl; two hours later the activation of neurons in the nucleus tractus solitarii (NTS) of the brainstem was visualized by c‐Fos immunocytochemistry. Mild gastritis was induced by addition of iodoacetamide (0.1%) to the drinking water for 7 days. Exposure of the gastric mucosa to HCl (0.25 M) caused a 3‐fold increase in the number of c‐Fos‐positive neurons in the NTS. This afferent input to the NTS remained unchanged by ASIC3 knockout, whereas ASIC2 knockout augmented the c‐Fos response to gastric HCl challenge by 33% (P < 0.01). Pretreatment of wild‐type mice with iodoacetamide induced mild gastritis, as revealed by increased myeloperoxidase activity, and enhanced the number of NTS neurons responding to gastric HCl challenge by 41% (P < 0.01). This gastric acid hyperresponsiveness was absent in ASIC3 knockout mice but fully preserved in ASIC2 knockout mice. The current data indicate that ASIC3 plays a major role in the acid hyperresponsiveness associated with experimental gastritis. In contrast, ASIC2 appears to dampen acid‐evoked input from the stomach to the NTS.

Human Stiff-Person Syndrome IgG Induces Anxious Behavior in Rats

Background Anxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxious behavior can occur in a variety of diseases. Stiff person syndrome (SPS) is a CNS disorder characterized by increased muscle tone and prominent agoraphobia and anxiety. Most patients have high-titer antibodies against glutamate decarboxylase (GAD) 65. The pathogenic role of these autoantibodies is unclear. Methodology/Principal Findings We re-investigated a 53 year old woman with SPS and profound anxiety for GABA-A receptor binding in the amygdala with (11)C-flumazenil PET scan and studied the potential pathogenic role of purified IgG from her plasma filtrates containing high-titer antibodies against GAD 65. We passively transferred the IgG fraction intrathecally into rats and analyzed the effects using behavioral and in vivo electrophysiological methods. In cell culture, we measured the effect of patient IgG on GABA release from hippocampal neurons. Repetitive intrathecal application of purified patient IgG in rats resulted in an anxious phenotype resembling the core symptoms of the patient. Patient IgG selectively bound to rat amygdala, hippocampus, and frontal cortical areas. In cultured rat hippocampal neurons, patient IgG inhibited GABA release. In line with these experimental results, the GABA-A receptor binding potential was reduced in the patients amygdala/hippocampus complex. No motor abnormalities were found in recipient rats. Conclusion/Significance The observations in rats after passive transfer lead us to propose that anxiety-like behavior can be induced in rats by passive transfer of IgG from a SPS patient positive for anti-GAD 65 antibodies. Anxiety, in this case, thus may be an antibody-mediated phenomenon with consecutive disturbance of GABAergic signaling in the amygdala region.

Gene–environment interaction influences anxiety-like behavior in ethologically based mouse models

Ethologically based animal models are widely used; however, results from different laboratories vary significantly which may partly be due to the lack of standardization. Here, we examined the effects of circadian rhythm, lighting condition and mouse strain (BALB/c and C57BL/6, known to differ in measures of avoidance and risk assessment behavior) on two well established behavioral tests in mice: the Elevated Plus Maze (EPM) and the Open Field (OF). Parameters from both paradigms are commonly used as indices of anxiety-like behavior. BALB/c mice and C57BL/6 mice were independently tested in the morning and at night, in regular laboratory lighting and in the dark. We developed a novel method based on infrared lighting from below, coupled to respective video-tracking equipment, which facilitates standard testing of behavior interference-free in complete darkness. The two mouse strains differed in anxiety-related variables for the EPM in the dark, and for the OF in regular laboratory lighting. Moreover, BALB/c displayed greater anxiety-like behavior than C57BL/6 in the OF but less anxiety-like behavior than C57BL/6 in the EPM. Lighting condition has a major influence on both behavioral tests and this to a considerably larger extent than circadian rhythm. In addition, the lighting condition interacts strongly with the genetic background, producing discriminative differences in the anxiety-related variables depending on mouse strain and lighting condition. These results challenge the comparability of not sufficiently standardized tests of anxiety-like behavior and emphasize the need for controlling environmental variables in behavioral phenotyping.

Decreased anxiety in mice lacking the organic cation transporter 3.

The organic cation transporter 3 (OCT3; synonymous: extraneuronal monoamine transporter, EMT, Slc22a3) encodes an isoform of the organic cation transporters and is expressed widely across the whole brain. OCTs are a family of high-capacity, bidirectional, multispecific transporters of organic cations. These also include serotonin, dopamine and norepinephrine making OCTs attractive candidates for a variety of neuropsychiatric disorders including anxiety disorders. OCT3 has been implicated in termination of monoaminergic signalling in the central nervous system. Interestingly, OCT3 mRNA is however also significantly up-regulated in the hippocampus of serotonin transporter knockout mice where it might serve as an alternative reuptake mechanism for serotonin. The examination of the behavioural phenotype of OCT3 knockout mice thus is paramount to assess the role of OCT3. We have therefore subjected mice lacking the OCT3 gene to a comprehensive behavioural test battery. While cognitive functioning in the Morris water maze test and aggression levels measured with the resident–intruder paradigm were in the same range as the respective control animals, OCT3 knockout animals showed a tendency of increased activity and were significantly less anxious in the elevated plus-maze test and the open field test as compared to their respective wild-type controls arguing for a role of OCT3 in the regulation of fear and anxiety, probably by modulating the serotonergic tone in limbic circuitries.

Experimental gastritis in mice enhances anxiety in a gender-related manner

There is a gender-related comorbidity of pain-related and inflammatory bowel diseases with psychiatric diseases. Since the impact of experimental gastrointestinal inflammation on the emotional-affective behavior is little known, we examined whether experimental gastritis modifies anxiety, stress coping and circulating corticosterone in male and female Him:OF1 mice. Gastritis was induced by adding iodoacetamide (0.1%) to the drinking water for at least 7 days. Inflammation was assessed by gastric histology and myeloperoxidase activity, circulating corticosterone determined by enzyme immunoassay, anxiety-related behavior evaluated with the elevated plus maze and stress-induced hyperthermia tests, and depression-like behavior estimated with the tail suspension test. Iodoacetamide-induced gastritis was associated with gastric mucosal surface damage and an increase in gastric myeloperoxidase activity, this increase being significantly larger in female mice than in male mice. The rectal temperature of male mice treated with iodoacetamide was enhanced, whereas that of female mice was diminished. The circulating levels of corticosterone were reduced by 65% in female mice treated with iodoacetamide but did not significantly change in male mice. On the behavioral level, iodoacetamide treatment caused a decrease in nocturnal home-cage activity, drinking and feeding. While depression-related behavior remained unaltered following induction of gastritis, behavioral indices of anxiety were significantly enhanced in female but not male mice. There was no correlation between the estrous cycle and anxiety as well as circulating corticosterone. Radiotracer experiments revealed that iodoacetamide did not readily enter the brain, the blood-brain ratio being 20:1. Collectively, these data show that iodoacetamide treatment causes gastritis in a gender-related manner, its severity being significantly greater in female than in male mice. The induction of gastritis in female mice is associated with a reduction of circulating corticosterone and an enforcement of behavioral indices of anxiety. Gastric inflammation thus has a distinct gender-dependent influence on emotional-affective behavior and its neuroendocrine control.

Human IgG directed against amphiphysin induces anxiety behavior in a rat model after intrathecal passive transfer

Stiff person syndrome with auto-antibodies against amphiphysin is characterized by muscular stiffness, spasms, and anxiety which is a less appreciated core symptom. Here, we report that intrathecal application of purified immunoglobulin G-antibodies against amphiphysin from one patient induce anxiety behavior in rats. Immunostaining demonstrated binding of anti-amphiphysin antibodies to brain structures which are associated with anxiety disorders, such as the amygdala. We propose that antibody-mediated amphiphysin deficiency may account for anxiety behavior in stiff person syndrome via presynaptic dysregulation of GABAergic pathways.

Endogenous neuropeptide Y depresses the afferent signaling of gastric acid challenge to the mouse brainstem via neuropeptide Y type Y2 and Y4 receptors.

Vagal afferents signal gastric acid challenge to the nucleus tractus solitarii of the rat brainstem. This study investigated whether nucleus tractus solitarii neurons in the mouse also respond to gastric acid challenge and whether this chemonociceptive input is modified by neuropeptide Y acting via neuropeptide Y receptors of type Y2 or Y4. The gastric mucosa of female mice was exposed to different concentrations of HCl or saline, excitation of neurons in the nucleus tractus solitarii visualized by c-Fos immunohistochemistry, gastric emptying deduced from the gastric volume recovery, and gastric lesion formation evaluated by planimetry. Relative to saline, intragastric HCl (0.15-0.35 M) increased the number of c-Fos-expressing cells in the nucleus tractus solitarii in a concentration-dependent manner, inhibited gastric emptying but failed to cause significant hemorrhagic injury in the stomach. Mice in which the Y2 or Y4 receptor gene had been deleted responded to gastric acid challenge with a significantly higher expression of c-Fos in the nucleus tractus solitarii, the increases amounting to 39 and 31%, respectively. The HCl-induced inhibition of gastric emptying was not altered by deletion of the Y2 or Y4 receptor gene. BIIE0246 ((S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6H)-oxodibenz[b,e] azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl] acetyl]-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide; 0.03 mmol/kg s.c.), a Y2 receptor antagonist which does not cross the blood-brain barrier, did not modify the c-Fos response to gastric acid challenge. The Y2 receptor agonist peptide YY-(3-36) (0.1 mg/kg intraperitoneally) likewise failed to alter the gastric HCl-evoked expression of c-Fos in the nucleus tractus solitarii. BIIE0246, however, prevented the effect of peptide YY-(3-36) to inhibit gastric acid secretion as deduced from measurement of intragastric pH. The current data indicate that gastric challenge with acid concentrations that do not induce overt injury but inhibit gastric emptying is signaled to the mouse nucleus tractus solitarii. Endogenous neuropeptide Y acting via Y2 and Y4 receptors depresses the afferent input to the nucleus tractus solitarii by a presumably central site of action.