Adipose-derived mesenchymal stem cells rescue rat hippocampal cells from aluminum oxide nanoparticle-induced apoptosis via regulation of P53, Aß, SOX2, OCT4, and CYP2E1.

Mesenchymal stem cells (MSCs) possess a preventive capacity against free radical toxicity in various tissues. The present study aimed to demonstrate the reformative and treatment roles of adipose-derived MSCs (AD-MSCs) against severe toxicity in the hippocampal cells of the brain caused by aluminum oxide nanoparticles (Al2O3-NPs). Rats were divided into five experimental groups: an untreated control group, a control group receiving NaCl, a group receiving Al2O3-NPs (6 mg/kg) for 20 days, a group that was allowed to recover (R) for 20 days following treatment with Al2O3-NPs, and a Al2O3-NPs + AD-MSCs group, where each rat was injected with 0.8 × 106 AD-MSCs via the caudal vein. Oral administration of Al2O3-NPs increased the protein levels of P53, cleaved caspase-3, CYP2E1, and beta-amyloid (Aß); contrarily, AD-MSCs transplantation downregulated the levels of these proteins. In addition, the AD-MSCs-treated hippocampal cells were protected from Al2O3-NPs-induced toxicity, as detected by the expression levels of Sox2 and Oct4 that are essential for the maintenance of self-renewal. It was also found that AD-MSCs injection significantly altered the levels of brain total peroxide and monoamine oxidase (MAO)-A and MAO-B activities. Histologically, our results indicated that AD-MSCs alleviated the severe damage in the hippocampal cells induced by Al2O3-NPs. Moreover, the role of AD-MSCs in reducing hippocampal cell death was reinforced by the regulation of P53, cleaved caspase-3, Aß, and CYP2E1 proteins, as well as by the regulation of SOX2 and OCT4 levels and MAO-A and MAO-B activities.

Ca2+-associated triphasic pH changes in mitochondria during brown adipocyte activation.

OBJECTIVE: Brown adipocytes (BAs) are endowed with a high metabolic capacity for energy expenditure due to their high mitochondria content. While mitochondrial pH is dynamically regulated in response to stimulation and, in return, affects various metabolic processes, how mitochondrial pH is regulated during adrenergic stimulation-induced thermogenesis is unknown. We aimed to reveal the spatial and temporal dynamics of mitochondrial pH in stimulated BAs and the mechanisms behind the dynamic pH changes. METHODS: A mitochondrial targeted pH-sensitive protein, mito-pHluorin, was constructed and transfected to BAs. Transfected BAs were stimulated by an adrenergic agonist, isoproterenol. The pH changes in mitochondria were characterized by dual-color imaging with indicators that monitor mitochondrial membrane potential and heat production. The mechanisms of pH changes were studied by examining the involvement of electron transport chain (ETC) activity and Ca2+ profiles in mitochondria and the intracellular Ca2+ store, the endoplasmic reticulum (ER). RESULTS: A triphasic mitochondrial pH change in BAs upon adrenergic stimulation was revealed. In comparison to a thermosensitive dye, we reveal that phases 1 and 2 of the pH increase precede thermogenesis, while phase 3, characterized by a pH decrease, occurs during thermogenesis. The mechanism of pH increase is partially related to ETC. In addition, the pH increase occurs concurrently with an increase in mitochondrial Ca2+. This Ca2+ increase is contributed to by an influx from the ER, and it is further involved in mitochondrial pH regulation. CONCLUSIONS: We demonstrate that an increase in mitochondrial pH is implicated as an early event in adrenergically stimulated BAs. We further suggest that this pH increase may play a role in the potentiation of thermogenesis.

Mitochondrial involvement in memory impairment induced by scopolamine in rats.

OBJECTIVE: Scopolamine (SCO) administration to rats induces molecular features of AD and other dementias, including impaired cognition, increased oxidative stress, and imbalanced cholinergic transmission. Although mitochondrial dysfunction is involved in different types of dementias, its role in cognitive impairment induced by SCO has not been well elucidated. The aim of this work was to evaluate the in vivo effect of SCO on different brain mitochondrial parameters in rats to explore its neurotoxic mechanisms of action. METHODS: Saline (Control) or SCO (1 mg/kg) was administered intraperitoneally 30 min prior to neurobehavioral and biochemical evaluations. Novel object recognition and Y-maze paradigms were used to evaluate the impact on memory, while redox profiles in different brain regions and the acetylcholinesterase (AChE) activity of the whole brain were assessed to elucidate the amnesic mechanism of SCO. Finally, the effects of SCO on brain mitochondria were evaluated both ex vivo and in vitro, the latter to determine whether SCO could directly interfere with mitochondrial function. RESULTS: SCO administration induced memory deficit, increased oxidative stress, and increased AChE activities in the hippocampus and prefrontal cortex. Isolated brain mitochondria from rats administered with SCO were more vulnerable to mitochondrial swelling, membrane potential dissipation, H2O2 generation and calcium efflux, all likely resulting from oxidative damage. The in vitro mitochondrial assays suggest that SCO did not affect the organelle function directly. CONCLUSION: In conclusion, the present results indicate that SCO induced cognitive dysfunction and oxidative stress may involve brain mitochondrial impairment, an important target for new neuroprotective compounds against AD and other dementias.

Evaluation of the open time of calcium channels at variation potential generation in wheat leaf cells.

The role of ions in the generation and mechanism of propagation of variation potential (VP) has been widely investigated. It is likely that Ca(2+) influx via calcium channels is an initial stage of VP; however, development of long-term membrane depolarization requires prolonged open times of calcium channels. We investigated depolarization time in the present study. It was shown that local burning induced VP in wheat seedling and the electrical response was suppressed under EGTA presence. Depolarization formation, which may indicate open time of calcium channels at VP generation, was observed up to 30 s after reaction induction when calcium ions were added to initially calcium-free medium. Long-term calcium channel open time may be the reason for long membrane depolarization at VP and may also be connected with the type of channels participating in wound reaction propagation.

Tetraarsenic oxide-induced inhibition of malignant glioma cell invasion in vitro via a decrease in matrix metalloproteinase secretion and protein kinase B phosphorylation.

OBJECT: Local invasiveness of malignant glioma is a major reason for the failure of current treatments including surgery and radiation therapy. Tetraarsenic oxide (As4O6 [TAO]) is a trivalent arsenic compound that has potential anticancer and antiangiogenic effects in selected cancer cell lines at a lower concentration than arsenic trioxide (As2O3 [ATO]), which has been more widely tested in vitro and in vivo. The authors tried to determine the cytotoxic concentration of TAO in malignant glioma cell lines and whether TAO would show anti-invasive effects under conditions independent of cell death or apoptosis. METHODS: The human phosphatase and tensin homolog (PTEN)-deficient malignant glioma cell lines U87MG, U251MG, and U373MG together with PTEN-functional LN428 were cultured with a range of micromolar concentrations of TAO. The invasiveness of the glioma cell lines was analyzed. The effect of TAO on matrix metalloproteinase (MMP) secretion and membrane type 1 (MT1)-MMP expression was measured using gelatin zymography and Western blot, respectively. Akt, or protein kinase B, activity, which is a downstream effector of PTEN, was assessed with a kinase assay using glycogen synthesis kinase-3ß (GSK-3ß) as a substrate and Western blotting of phosphorylated Akt. RESULTS: Tetraarsenic oxide inhibited 50% of glioma cell proliferation at 6.3-12.2 µM. Subsequent experiments were performed under the same TAO concentrations and exposure times, avoiding the direct tumoricidal effect of TAO, which was confirmed with apoptosis markers. An invasion assay revealed a dose-dependent decrease in invasiveness under the influence of TAO. Both the gelatinolytic activity of MMP-2 and MT1-MMP expression decreased in a dose-dependent manner in all cell lines, which was in accordance with the invasion assay results. The TAO decreased kinase activity of Akt on GSK-3ß assay and inhibited Akt phosphorylation in a dose-dependent manner in all cell lines regardless of their PTEN status. CONCLUSIONS: These results showed that TAO effectively inhibits proliferation of glioblastoma cell lines and also exerts an anti-invasive effect via decreased MMP-2 secretion, decreased MT1-MMP expression, and the inhibition of Akt phosphorylation under conditions devoid of cytotoxicity. Further investigations using an in vivo model are needed to evaluate the potential role of TAO as an anti-invasive agent.

EF-hand motifs of diacylglycerol kinase α interact intra-molecularly with its C1 domains.

Diacylglycerol kinase (DGK) α, which is activated by Ca(2+), contains a recoverin homology (RVH) domain, tandem repeats of two Ca(2+)-binding EF-hand motifs, two cysteine-rich C1 domains and the catalytic domain. We previously found that a DGKα mutant lacking the RVH domain and EF-hands was constitutively active and that the N-terminal region of DGKα, consisting of the RVH domain and EF-hand motifs, interacted intra-molecularly with the C-terminal region containing the C1 and catalytic domains. In this study, we narrowed down the interaction regions of DGKα. At the C-terminal region, the C1 domains are responsible for the intra-molecular interaction. At the N-terminal region, the EF-hand motifs mainly contribute to the interaction. Moreover, using highly purified EF-hand motifs and C1 domains, we demonstrate that they directly bind to each other. The co-precipitation of these two domains was clearly attenuated by the addition of Ca(2+). These results indicate that the Ca(2+)-induced dissociation of the intra-molecular interaction between the EF-hand motifs and the C1 domains of DGKα is the key event that regulates the activity of the enzyme.

Microglial NADPH oxidase activation mediates rod cell death in the retinal degeneration in rd mice.

Accumulating evidence supports that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contributes to microglia-mediated neurotoxicity in the CNS neurodegenerative diseases. Several studies, including ours, suggest that microglial activation is involved in the retinal degeneration in the animal models of retinitis pigmentosa (RP). In the present study, we investigated the activation of NADPH oxidase in the rod degeneration in rd mice and further explored its role in the microglia-mediated photoreceptor apoptosis. Expression of gp91phox protein, a major subunit of NAPDH oxidase in the whole retina of rd mice at postnatal days (P) 8, 10, 12, 14, 16 and 18 was assessed by western blot analysis. Location of gp91phox in the rd retina at each age group and its cellular source were studied by immunohistochemical analysis and double labeling respectively. The generation of superoxide radicals in the rd retinas was demonstrated by intraperitoneal injection of hydroethidine. Apocynin was applied intraperitoneally in the rd mice from P8 to P14 to inhibit the activity of NAPDH oxidase and the outer nuclear layer (ONL) thickness was measured before and after apocynin treatment. Our results demonstrated that during the rod degenerative process, the expression of gp91phox started to increase in the outer part of rd retina at P10 and reached a peak at P14. Double labeling of gp91phox with CD11b showed co-localization of gp91phox in the retinal microglial cells. Increasing generation of superoxide radicals visualized by hydroethidine was noted at P8 and reached a peak at P14. Apocynin markedly reduced the production of superoxide radicals and preserved the rod cells. The results suggested that NADPH oxidase might play an important role in the rod degeneration in the rd mice. Inhibition of NAPDH oxidase could be a possible approach to treat RP in the early degenerative stage.

Purinergic signaling negatively regulates activity of an olfactory receptor in an odorant-dependent manner.

Extracellular purines and pyrimidines are important signaling molecules that mediate diverse biological functions via cell surface purinergic receptors. Although purinergic modulation to olfactory activity has been reported, cell-specific expression and action of purinergic receptors deserve further exploration. We physiologically characterized expression of purinergic receptors in a set of olfactory sensory neurons that are responsive to both acetophenone and benzaldehyde (AB-OSNs). Sparsely distributed in the most ventral olfactory receptor zone, AB-OSNs were activated by P2 purinergic receptor agonists but not by P1 purinergic receptor agonist adenosine. Both P2X-selective agonist α,ß-methylene ATP and P2Y-selective agonist uridine 5'-triphosphate (UTP) were stimulatory to AB-OSNs, indicating expression of both P2X and P2Y purinergic receptors in AB-OSNs. Pharmacological characterization of receptor specificity using various P2X and P2Y agonists and antagonists illustrated that P2X1 and P2Y2 receptors played major roles in purinergic signaling in AB-OSNs. Interestingly, the results of purinergic modulation to acetophenone-evoked responses were different from those to benzaldehyde-evoked responses within the same neurons. Activation of P2X1 receptors had more profound inhibitory effects on benzaldehyde-evoked intracellular calcium elevation than on acetophenone-evoked responses within the same neurons, and the reverse was true when P2Y2 receptors were activated. Cross-adaptation data showed that acetophenone and benzaldehyde bound to the same olfactory receptor. Thus, our study has demonstrated that purinergic signaling of P2X and P2Y receptors has different effects on olfactory transduction mediated by a defined olfactory receptor and the consequences of purinergic modulation of olfactory activity might depend on stereotypic structures of the odorant-receptor complex.

Early modulation of the transcription factor Nrf2 in rodent striatal slices by quinolinic acid, a toxic metabolite of the kynurenine pathway.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor involved in the orchestration of antioxidant responses. Although its pharmacological activation has been largely hypothesized as a promising tool to ameliorate the progression of neurodegenerative events, the actual knowledge about its modulation in neurotoxic paradigms remains scarce. In this study, we investigated the early profile of Nrf2 modulation in striatal slices of rodents incubated in the presence of the toxic kynurenine pathway metabolite, quinolinic acid (QUIN). Tissue slices from rats and mice were obtained and used throughout the experiments in order to compare inter-species responses. Nuclear Nrf2 protein levels and oxidative damage to lipids were compared. Time- and concentration-response curves of all markers were explored. Nrf2 nuclear activation was corroborated through phase 2 antioxidant protein expression. The effects of QUIN on Nrf2 modulation and oxidative stress were also compared between slices of wild-type (Nrf2(+/+)) and Nrf2 knock-out (Nrf2(-/-)) mice. The possible involvement of the N-methyl-d-aspartate receptor (NMDAr) in the Nrf2 modulation and lipid peroxidation was further explored in mice striatal slices. In rat striatal slices, QUIN stimulated the Nrf2 nuclear translocation. This effect was accompanied by augmented lipid peroxidation. In the mouse striatum, QUIN per se exerted an induction of Nrf2 factor only at 1h of incubation, and a concentration-response effect on lipid peroxidation after 3h of incubation. QUIN stimulated the striatal content of phase 2 enzymes. Nrf2(-/-) mice were slightly more responsive than Nrf2(+/+) mice to the QUIN-induced oxidative damage, and completely unresponsive to the NMDAr antagonist MK-801 when tested against QUIN. Findings of this study indicate that: (1) Nrf2 is modulated in rodent striatal tissue in response to QUIN; (2) Nrf2(-/-) striatal tissue was moderately more vulnerable to oxidative damage than the Wt condition; and (3) early Nrf2 up-regulation reflects a compensatory response to the QUIN-induced oxidative stress in course as part of a general defense system, whereas Nrf2 down-regulation might contribute to more intense oxidative cell damage.

Changes in immune cell populations in the periphery and liver of GBV-B-infected and convalescent tamarins (Saguinus labiatus).

Flaviviruses related to hepatitis C virus (HCV) in suitable animal models may provide further insight into the role that cellular immunity contributes to spontaneous clearance of HCV. We characterised changes in lymphocyte populations in tamarins with an acute GBV-B infection, a hepatitis virus of the flaviviridae. Major immune cell populations were monitored in peripheral and intra-hepatic lymphocytes at high viraemia or following a period when peripheral virus was no longer detected. Limited changes in major lymphocyte populations were apparent during high viraemia; however, the proportions of CD3(+) lymphocytes decreased and CD20(+) lymphocytes increased once peripheral viraemia became undetectable. Intrahepatic lymphocyte populations increased at both time points post-infection. Distinct expression patterns of PD-1, a marker of T-cell activation, were observed on peripheral and hepatic lymphocytes; notably there was elevated PD-1 expression on hepatic CD4(+) T-cells during high viraemia, suggesting an activated phenotype, which decreased following clearance of peripheral viraemia. At times when peripheral vRNA was not detected, suggesting viral clearance, we were able to readily detect GBV-B RNA in the liver, indicative of long-term virus replication. This study is the first description of changes in lymphocyte populations during GBV-B infection of tamarins and provides a foundation for more detailed investigations of the responses that contribute to the control of GBV-B infection.

The cholinergic agonist carbachol increases the frequency of spontaneous GABAergic synaptic currents in dorsal raphe serotonergic neurons in the mouse.

Dorsal raphe nucleus (DRN) serotonin (5-HT) neurons play an important role in feeding, mood control and stress responses. One important feature of their activity across the sleep-wake cycle is their reduced firing during rapid-eye-movement (REM) sleep which stands in stark contrast to the wake/REM-on discharge pattern of brainstem cholinergic neurons. A prominent model of REM sleep control posits a reciprocal interaction between these cell groups. 5-HT inhibits cholinergic neurons, and activation of nicotinic receptors can excite DRN 5-HT neurons but the cholinergic effect on inhibitory inputs is incompletely understood. Here, in vitro, in DRN brain slices prepared from GAD67-GFP knock-in mice, a brief (3 min) bath application of carbachol (50 µM) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in GFP-negative, putative 5-HT neurons but did not affect miniature (tetrodotoxin-insensitive) IPSCs. Carbachol had no direct postsynaptic effect. Thus, carbachol likely increases the activity of local GABAergic neurons which synapse on 5-HT neurons. Removal of dorsal regions of the slice including the ventrolateral periaqueductal gray (vlPAG) region where GABAergic neurons projecting to the DRN have been identified, abolished the effect of carbachol on sIPSCs whereas the removal of ventral regions containing the oral region of the pontine reticular nucleus (PnO) did not. In addition, carbachol directly excited GFP-positive, GABAergic vlPAG neurons. Antagonism of both muscarinic and nicotinic receptors completely abolished the effects of carbachol. We suggest cholinergic neurons inhibit DRN 5-HT neurons when acetylcholine levels are lower i.e. during quiet wakefulness and the beginning of REM sleep periods, in part via excitation of muscarinic and nicotinic receptors located on local vlPAG and DRN GABAergic neurons. Higher firing rates or burst firing of cholinergic neurons associated with attentive wakefulness or phasic REM sleep periods leads to excitation of 5-HT neurons via the activation of nicotinic receptors located postsynaptically and presynaptically on excitatory afferents.

Caspase inhibition augments Dichlorvos-induced dopaminergic neuronal cell death by increasing ROS production and PARP1 activation.

Numerous epidemiological studies have shown an association between pesticide exposure and the increased risk of developing Parkinson's disease. Previously we have reported that Dichlorvos exposure can induce oxidative stress, resulting in over-expression of pro-apoptotic genes and finally caspase-dependent nigrostriatal dopaminergic neuronal cell death in rat brain. Here, we examined the effect of caspase inhibition on PC12 cell death induced by Dichlorvos (30 µM). Reactive oxygen species (ROS) generation followed by protein carbonylation, lipid peroxidation, decreased antioxidant defenses (decreased Mn-superoxide dismutase (MnSOD) activity and decreased glutathione levels) and subsequent caspase activation mediated the apoptosis. Inhibition of caspase cascade with Boc-aspartyl(OMe)-fluoromethylketone (BAF) enhanced the Dichlorvos-induced PC12 cell death, as assessed by the increased cellular efflux of lactate dehydrogenase (LDH). This increase in cell death was accompanied by a marked increase in poly(ADP-ribose) polymerase-1 (PARP1) activity, increased oxidative stress, a reduction in the mitochondrial membrane potential and reduced cellular NAD and ATP levels. Pretreatment of cells with PJ34, a PARP1 inhibitor prevented the cells from undergoing cell death and preserved intracellular NAD and ATP levels. Subsequent release of the apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus was also prevented by PJ34 pretreatment. In conclusion, the results of the present study show that caspase inhibition without concurrent inhibition of PARP1 is unlikely to be effective in preventing cell death because in the presence of the caspase inhibitor, caspase-independent cell death predominates due to PARP activation. These results suggest that combined therapeutic strategies directed at multiple cell death pathways may provide superior neuroprotection than those directed at a single mechanism.

Quantification of the density of cooperative neighboring synapses required to evoke endocannabinoid signaling.

The spatial pattern of synapse activation may impact on synaptic plasticity. This applies to the synaptically-evoked endocannabinoid-mediated short-term depression at the parallel fiber (PF) to Purkinje cell synapse, the occurrence of which requires close proximity between the activated synapses. Here, we determine quantitatively this required proximity, helped by the geometrical organization of the cerebellar molecular layer. Transgenic mice expressing a calcium indicator selectively in granule cells enabled the imaging of action potential-evoked presynaptic calcium rise in isolated, single PFs. This measurement was used to derive the number of PFs activated within a beam of PFs stimulated in the molecular layer, from which the density of activated PFs (input density) was calculated. This density was on average 2.8 µm(-2) in sagittal slices and twice more in transverse slices. The synaptically-evoked endocannabinoid-mediated suppression of excitation (SSE) evoked by ten stimuli at 200 Hz was determined from the monitoring of either postsynaptic responses or presynaptic calcium rise. The SSE was significantly larger when recorded in transverse slices, where the input density is larger. The exponential description of the SSE plotted as a function of the input density suggests that the SSE is half reduced when the input density decreases from 6 to 2 µm(-2). We conclude that, although all PFs are truncated in an acute sagittal slice, half of them remain respondent to stimulation, and activated synapses need to be closer than 1.5 µm to synergize in endocannabinoid signaling.

Genetic deficiency of ß2-containing nicotinic receptors attenuates brain injury in ischemic stroke.

One of the major consequences of stroke is brain injury caused by glutamate-mediated excitotoxicity. Glutamate-mediated excitatory activities are partially driven by ß2-containing nicotinic acetylcholine receptors (ß2-nAChRs). In examining the role of ß2-nAChRs in cerebral ischemic injury, excitotoxicity and stroke outcome, we found that deficiency of ß2-nAChRs attenuated brain infarction and neurological deficit at 24 and 72 h after transient middle cerebral artery occlusion (MCAO). Genetic deletion of ß2-nAChRs associated with reduced terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL(+)) and cleaved caspase-3(+) cells after MCAO, together with a reduction of extracellular glutamate and oxygen-glucose deprivation-induced increase of excitatory inputs in cortical neurons. Pharmacologic pretreatment with a selective ß2-nAChRs antagonist reduced brain infarction, neurological deficit, and MCAO-induced glutamate release. These findings suggest that deficiency of ß2-nAChRs, also achievable by pharmacological blockade, can decrease brain infarction and improve the neurological status in ischemic stroke. The improved outcome is associated with reduced extracellular glutamate level and lower excitatory inputs into ischemic neurons, suggesting a reduction of glutamate-mediated excitotoxicity in the mechanisms of neuroprotection.

Heterogeneous intrinsic excitability of murine spiral ganglion neurons is determined by Kv1 and HCN channels.

The spiral ganglion conveys afferent auditory information predominantly through a single class of type I neurons that receive signals from inner hair cell sensory receptors. These auditory primary afferents, like in other systems (Puopolo and Belluzzi, 1998; Gascon and Moqrich, 2010; Leao et al., 2012) possess a marked diversity in their electrophysiological features (Taberner and Liberman, 2005). Consistent with these observations, when the auditory primary afferents were assessed in neuronal explants separated from their peripheral and central targets it was found that individual neurons were markedly heterogeneous in their endogenous electrophysiological features. One aspect of this heterogeneity, obvious throughout the ganglion, was their wide range of excitability as assessed by voltage threshold measurements (Liu and Davis, 2007). Thus, while neurons in the base differed significantly from apical and middle neurons in their voltage thresholds, each region showed distinctly wide ranges of values. To determine whether the resting membrane potentials (RMPs) of these neurons correlate with the threshold distribution and to identify the ion channel regulatory elements underlying heterogeneous neuronal excitability in the ganglion, patch-clamp recordings were made from postnatal day (P5-8) murine spiral ganglion neurons in vitro. We found that RMP mirrored the tonotopic threshold distribution, and contributed an additional level of heterogeneity in each cochlear location. Pharmacological experiments further indicated that threshold and RMP was coupled through the Kv1 current, which had a dual impact on both electrophysiological parameters. Whereas, hyperpolarization-activated cationic channels decoupled these two processes by primarily affecting RMP without altering threshold level. Thus, beyond mechanical and synaptic specializations, ion channel regulation of intrinsic membrane properties imbues spiral ganglion neurons with different excitability levels, a feature that contributes to primary auditory afferent diversity.

GABAergic influence on temporomandibular joint-responsive spinomedullary neurons depends on estrogen status.

Sensory input from the temporomandibular joint (TMJ) to neurons in superficial laminae at the spinomedullary (Vc/C1-2) region is strongly influenced by estrogen status. This study determined if GABAergic mechanisms play a role in estrogen modulation of TMJ nociceptive processing in ovariectomized female rats treated with high- (HE) or low-dose (LE) estradiol (E2) for 2days. Superficial laminae neurons were activated by ATP (1mM) injections into the joint space. The selective GABAA receptor antagonist, bicuculline methiodide (BMI, 5 or 50µM, 30µl), applied at the site of recording greatly enhanced the magnitude and duration of ATP-evoked responses in LE rats, but not in units from HE rats. The convergent cutaneous receptive field (RF) area of TMJ neurons was enlarged after BMI in LE but not HE rats, while resting discharge rates were increased after BMI independent of estrogen status. By contrast, the selective GABAA receptor agonist, muscimol (50µM, 30µl), significantly reduced the magnitude and duration of ATP-evoked activity, resting discharge rate, and cutaneous RF area of TMJ neurons in LE and HE rats, whereas lower doses (5µM) affected only units from LE rats. Protein levels of GABAA receptor ß3 isoform at the Vc/C1-2 region were similar for HE and LE rats. These results suggest that GABAergic mechanisms contribute significantly to background discharge rates and TMJ-evoked input to superficial laminae neurons at the Vc/C1-2 region. Estrogen status may gate the magnitude of GABAergic influence on TMJ neurons at the earliest stages of nociceptive processing at the spinomedullary region.

Regional and genotypic differences in intrinsic electrophysiological properties of cerebellar Purkinje neurons from wild-type and dystrophin-deficient mdx mice.

Cerebellar subregions are recognized as having specialized roles, with lateral cerebellum considered crucial for cognitive processing, whereas vermal cerebellum is more strongly associated with motor control. In human Duchenne muscular dystrophy, loss of the cytoskeletal protein dystrophin is thought to cause impairments in cognition, including learning and memory. Previous studies demonstrate that loss of dystrophin causes dysfunctional signaling at γ-aminobutyric acid (GABA) synapses on Purkinje neurons, presumably by destabilization of GABAA receptors. However, potential differences in the intrinsic electrophysiological properties of Purkinje neurons, including membrane potential and action potential firing rates, have not been investigated. Here, using a 2×2 analysis of variance (ANOVA) experimental design, we employed patch clamp analysis to compare membrane properties and action potentials generated by acutely dissociated Purkinje neurons from vermal and lateral cerebellum in wild-type (WT) mice and mdx dystrophin-deficient mice. Compared to Purkinje neurons from WT mice, neurons from mdx mice exhibited more irregular action potential firing and a hyperpolarization of the membrane potential. Firing frequency was also lower in Purkinje neurons from the lateral cerebellum of mdx mice relative to those from WT mice. Several action potential waveform parameters differed between vermal and lateral Purkinje neurons, irrespective of dystrophin status, including action potential amplitude, slope (both larger in the vermal region), and duration (shorter in the vermal region). Moreover, the membrane potential of Purkinje neurons from the vermal region of WT mice exhibited a significant hyperpolarization and concurrent reduction in the frequency of spontaneous action potentials compared to Purkinje neurons from the lateral region. This regional hyperpolarization and reduction in spontaneous action potential frequency was abolished in mdx mice. These results from mice demonstrate the presence of differential electrophysiological properties between Purkinje neurons from different regions of the WT mouse cerebellum and altered intrinsic membrane properties in the absence of dystrophin. These findings provide a possible mechanism for the observations that absence of cerebellar dystrophin contributes to deficits in mental function observed in humans and mouse models of muscular dystrophy. Moreover, these results highlight the importance of distinguishing functional zones of the cerebellum in future work characterizing Purkinje neuron electrophysiology and studies using the model of dissociated Purkinje neurons from mice.

Dopaminergic enhancement of excitatory synaptic transmission in layer II entorhinal neurons is dependent on D1-like receptor-mediated signaling.

The modulatory neurotransmitter dopamine induces concentration-dependent changes in synaptic transmission in the entorhinal cortex, in which high concentrations of dopamine suppress evoked excitatory postsynaptic potentials (EPSPs) and lower concentrations induce an acute synaptic facilitation. Whole-cell current-clamp recordings were used to investigate the dopaminergic facilitation of synaptic responses in layer II neurons of the rat lateral entorhinal cortex. A constant bath application of 1 µM dopamine resulted in a consistent facilitation of EPSPs evoked in layer II fan cells by layer I stimulation; the size of the facilitation was more variable in pyramidal neurons, and synaptic responses in a small group of multiform neurons were not modulated by dopamine. Isolated inhibitory synaptic responses were not affected by dopamine, and the facilitation of EPSPs was not associated with a change in paired-pulse facilitation ratio. Voltage-clamp recordings of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptor-mediated excitatory postsynaptic currents (EPSCs) were facilitated by dopamine, but N-methyl-D-aspartate receptor-mediated currents were not. Bath application of the dopamine D1-like receptor blocker SCH23390 (50 µM), but not the D2-like receptor blocker sulpiride (50 µM), prevented the facilitation, indicating that it is dependent upon D1-like receptor activation. Dopamine D1 receptors lead to activation of protein kinase A (PKA), and including the PKA inhibitor H-89 or KT 5720 in the recording pipette solution prevented the facilitation of EPSCs. PKA-dependent phosphorylation of inhibitor 1 or the dopamine- and cAMP-regulated protein phosphatase (DARPP-32) can lead to a facilitation of AMPA receptor responses by inhibiting the activity of protein phosphatase 1 (PP1) that reduces dephosphorylation of AMPA receptors, and we found here that inhibition of PP1 occluded the facilitatory effect of dopamine. The dopamine-induced facilitation of AMPA receptor-mediated synaptic responses in layer II neurons of the lateral entorhinal cortex is therefore likely mediated via a D1 receptor-dependent increase in PKA activity and a resulting inhibition in PP1-dependent dephosphorylation of AMPA receptors.

Regulation of 3ß-hydroxysteroid dehydrogenase and sulphotransferase 2A1 gene expression in primary porcine hepatocytes by selected sex-steroids and plant secondary metabolites from chicory (Cichorium intybus L.) and wormwood (Artemisia sp.).

In pigs the endogenously produced compound androstenone is metabolised in the liver in two steps by 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and sulphotransferase 2A1 (SULT2A1). The present study investigated the effect of selected sex-steroids (0.01-1 µM androstenone, testosterone and estradiol), skatole (1-100 µM) and secondary plant metabolites (1-100 µM) on the expression of 3ß-HSD and SULT2A1 mRNA. Additionally the effect of a global methanolic extract of dried chicory root was investigated and compared to previous obtained in vivo effects. Primary hepatocytes were isolated from the livers of piglets (crossbreed: Landrace×Yorkshire and Duroc) and cultured for 24h before treatment for an additionally 24h. RNA was isolated from the hepatocytes and specific gene expression determined by RT-PCR using TaqMan probes. The investigated sex-steroids had no effect on the mRNA expression of 3ß-HSD and SULT2A1, while skatole decreased the content of SULT2A1 30% compared to control. Of the investigated secondary plant metabolites artemisinin and scoparone (found in Artemisia sp.) lowered the content of SULT2A1 by 20 and 30% compared to control, respectively. Moreover, we tested three secondary plant metabolites (lactucin, esculetin and esculin) found in chicory root. Lactucin increased the mRNA content of both 3ß-HSD and SULT2A1 by 200% compared to control. An extract of chicory root was shown to decrease the expression of both 3ß-HSD and SULT2A1. It is concluded that the gene expression of enzymes with importance for androstenone metabolism is regulated by secondary plant metabolites in a complex manner.

Vasopressin indirectly excites dorsal raphe serotonin neurons through activation of the vasopressin1A receptor.

The neuropeptide vasopressin (AVP; arginine-vasopressin) is produced in a handful of brain nuclei located in the hypothalamus and extended amygdala and is released both peripherally as a hormone and within the central nervous system as a neurotransmitter. Central projections have been associated with a number of functions including regulation of physiological homeostasis, control of circadian rhythms, and modulation of social behavior. The AVP neurons located in the bed nucleus of the stria terminalis and medial amygdala (i.e., extended amygdala) in particular have been associated with affiliative social behavior in multiple species. It was recently demonstrated that in the mouse AVP projections emanating from extended amygdala neurons innervate a number of forebrain and midbrain brain regions including the dorsal raphe nucleus (DR), the site of origin of most forebrain-projecting serotonin neurons. Based on the presence of AVP fibers in the DR, we hypothesized that AVP would alter the physiology of serotonin neurons via AVP 1A receptor (V1AR) activation. Using whole-cell electrophysiology techniques, we found that AVP increased the frequency and amplitude of excitatory post-synaptic currents (EPSCs) in serotonin neurons of male mice. The indirect stimulation of serotonin neurons was AMPA/kainate receptor dependent and blocked by the sodium channel blocker tetrodotoxin, suggesting an effect of AVP on glutamate neurons. Further, the increase in EPSC frequency induced by AVP was blocked by selective V1AR antagonists. Our data suggest that AVP had an excitatory influence on serotonin neurons. This work highlights a new target (i.e., V1AR) for manipulating serotonin neuron excitability. In light of our data, we propose that some of the diverse effects of AVP on physiology and behavior, including social behavior, may be due to activation of the DR serotonin system.