Adhesion to pharyngeal epithelium and modulation of immune response: Lactobacillus salivarius AR809, a potential probiotic strain isolated from the human oral cavity.

Microbiome modulators such as probiotics are known to modulate oral diseases. Very few probiotics are commercially available for use in the oral cavity. In this context, we selected human-origin Lactobacillus salivarius AR809 as a promising oropharyngeal probiotic and characterized its functional and immunomodulatory properties. Results demonstrated that AR809 could efficiently adhere to pharyngeal epithelial FaDu cells, antagonize Staphylococcus aureus, adapt to the oral environment, and modulate host innate immunity by inducing potentially protective effects. Particularly, AR809 diminished proinflammatory activity by enhancing the production of IL10 and inhibiting the expression of tumor necrosis factor-α, IL1B, inducible nitric oxide synthase, and RELA. Finally, we observed that AR809 grew efficiently when cultured in milk, suggesting that the preparation of a fermented milk product containing AR809 could be a practical way to administer this probiotic to humans. In conclusion, AR809 has high potential to adhere to the pharyngeal mucosa and could be applied in novel milk-based probiotic fermented food products.

ß-Catenin is required for maintaining hippocampal morphology during the perinatal period.

In mice, the compact hippocampal primordium is formed during the prenatal stage by early-generated neurons that migrate from the lateral ventricular zone. However, despite much being understood about the formation of the hippocampus, the molecular mechanisms that maintain the morphology of the hippocampal primordium after its formation remain to be characterized. ß-Catenin is a key factor of canonical Wnt signaling and also a component of adherens junctions. Previous embryonic deletion studies have demonstrated that ß-catenin is required for early development and generation of granule cells. However, whether ß-catenin is involved in the morphological maintenance of the hippocampus as a cell adhesion molecule is still unknown. Here, we report that perinatal deletion of ß-catenin in postmitotic neurons and some radial glial cells of hippocampus using CamKIIα-iCre; ß-cateninflox/flox conditional knockout mice, leads to disorganization of the radial glial scaffold and consequentially severe defects in hippocampal morphology. We demonstrate that ß-catenin is required for maintaining radial glial scaffold possibly via its well-known role in cell adhesion during the perinatal period. These findings provide essential advances into our understanding of the maintenance of the hippocampal primordium during the perinatal period.

Expression and regulated nuclear transport of transducers of regulated CREB 1 in retinal ganglion cells.

Calcium- and cAMP-dependent activation of CREB and transcription of cAMP-responsive element (CRE)-target genes play critical roles in various physiological and pathological conditions. TORCs (transducers of regulated CREB) represent a new family of conserved CREB coactivators that function as intracellular calcium- and cAMP-sensitive coincidence detectors, controlling the kinetics of CRE-mediated responses and long-term potentiation of synaptic transmission. Here we examined the expression and activity-dependent translocation of TORCs in adult retinal ganglion cells (RGCs), the primary target of acute retinal ischemic injury as well as chronic retinal degenerative diseases. We found that both mRNAs of TORC1 and TORC2, but not TORC3, were enriched in adult rat retina. Comparing with TORC2, TORC1 protein was highly and selectively expressed in RGCs. At resting condition, TORC1 protein was localized in the cytoplasm but not nucleus of RGCs. Activation of N-methyl-D-aspartate (NMDA) receptors by intravitreous injection of NMDA or increase of cAMP signaling by administration of forskolin triggered nuclear accumulation of TORC1. Furthermore, transient retinal ischemic injury resulted in peri-nuclear and nuclear accumulation of TORC1 as well as transcription of BDNF in RGCs. Our results demonstrate that TORC1 is enriched in RGCs and its subcellular location could be regulated by Ca(2+) and cAMP, suggesting that manipulation of TORC1 activity may promote survival of RGCs in some optic disease conditions.

Prolonged bursts occur in normal calcium in hippocampal slices after raising excitability and blocking synaptic transmission.

This study examined the conditions that are required for the appearance of the long-duration seizure-like activity that can be recorded in hippocampal slices. Spontaneous interictal activity was induced in CA1 and CA3 by perfusing hippocampal slices with high potassium, cesium, 4-aminopyridine, or tetraethylammonium chloride, in normal levels of calcium. Synaptic transmission was then blocked by the addition of neurotransmitter receptor blockers (6-cyano-7-nitroquinoxaline-2,3-dione, D,L-2-amino-5-phosphonopentanoic acid, and bicuculline) or the calcium channel blocker cadmium, resulting in complete blockade of the interictal discharges and the appearance of spontaneous seizure-like events (ictal-like discharges) primarily in CA1 and the dentate gyrus. Blocking synaptic transmission in normal artificial cerebrospinal fluid did not induce ictal-like discharges in any region. The results demonstrate that ictal-like discharges can appear in normal levels of extracellular calcium when chemical synaptic transmission is blocked pharmacologically. The results suggest that an increase in neuronal excitability and absence of interictal activity promote the appearance of the longer ictal-like discharges.

Effects of postsynaptic GABA(B) receptor activation on epileptiform activity in hippocampal slices.

GABA(B) receptor agonists have been reported to have both pro- and antiepileptic properties. Here, the effects of a GABA(B) receptor agonist, baclofen, were studied on epileptiform activity induced in the absence of synaptic transmission - to focus on the postsynaptic effects. Perfusion of hippocampal slices with 0-added calcium and high potassium induced field bursts in CA1 and the dentate gyrus. Addition of baclofen caused a transient suppression of the field bursts in CA1 and the dentate gyrus. The duration of the suppression was dependent on the concentration of baclofen and when the bursts reappeared they had a larger amplitude than before baclofen. Baclofen also suppressed the multiple population spikes evoked by antidromic stimulation in the dentate gyrus. This effect also decreased with continued baclofen perfusion. The effects of baclofen on the amplitude of the spontaneous field bursts and on the stimulation-induced multiple population spikes were blocked by the GABA(B) receptor antagonist SCH 50911, suggesting that these effects of baclofen are mediated by GABA(B) receptor activation. Baclofen significantly increased the peak extracellular K(+) concentration during each field burst in the dentate gyrus but did not change the baseline level of K(+) between field bursts. The results suggest that postsynaptic GABA(B) receptor activation by baclofen has transient antiepileptic effects followed by a rebound increase in excitability.

Regulation of extracellular pH in the developing hippocampus.

The increased propensity of the immature brain to have seizures may be due, in part, to an inability to regulate the extracellular ionic environment. Here, we tested the regulation of extracellular pH in the developing hippocampus during and after intense neuronal activity induced by 20 Hz electrical stimulation in vivo. In CA1, stimulus trains to the CA3 region elicited an early alkaline shift associated with a later slow acid shift in all ages tested (2 weeks old, 3 weeks old and adult). In the dentate gyrus in adults, stimulation only elicited acidification. An initial alkalinization was only observed in the dentate gyrus in 2-week-old animals. The rate of recovery of the extracellular pH after termination of the stimulation was slower in the younger animals compared to adults in both CA1 and the dentate gyrus. The results indicate that the extracellular pH is regulated by mechanisms that undergo developmental changes that parallel the development of the dentate gyrus and development of the regulation of extracellular potassium.

pH Sensitivity of non-synaptic field bursts in the dentate gyrus.

Under conditions of low [Ca(2+)](o) and high [K(+)](o), the rat dentate granule cell layer in vitro develops recurrent spontaneous prolonged field bursts that resemble an in vivo phenomenon called maximal dentate activation. To understand how pH changes in vivo might affect this phenomenon, the slices were exposed to different extracellular pH environments in vitro. The field bursts were highly sensitive to extracellular pH over the range 7.0-7.6 and were suppressed at low pH and enhanced at high pH. Granule cell resting membrane potential, action potentials, and postsynaptic potentials were not significantly altered by pH changes within the range that suppressed the bursts. The pH sensitivity of the bursts was not altered by pharmacologic blockade of N-methyl-D-aspartate (NMDA), non-NMDA, and GABA(A) receptors at concentrations of these agents sufficient to eliminate both spontaneous and evoked synaptic potentials. Gap junction patency is known to be sensitive to pH, and agents that block gap junctions, including octanol, oleamide, and carbenoxolone, blocked the prolonged field bursts in a manner similar to low pH. Perfusion with gap junction blockers or acidic pH suppressed field bursts but did not block spontaneous firing of single and multiple units, including burst firing. These data suggest that the pH sensitivity of seizures and epileptiform phenomena in vivo may be mediated in large part through mechanisms other than suppression of NMDA-mediated or other excitatory synaptic transmission. Alterations in electrotonic coupling via gap junctions, affecting field synchronization, may be one such process.

Extracellular pH responses in CA1 and the dentate gyrus during electrical stimulation, seizure discharges, and spreading depression.

Since neuronal excitability is sensitive to changes in extracellular pH and there is regional diversity in the changes in extracellular pH during neuronal activity, we examined the activity-dependent extracellular pH changes in the CA1 region and the dentate gyrus. In vivo, in the CA1 region, recurrent epileptiform activity induced by stimulus trains, bicuculline, and kainic acid resulted in biphasic pH shifts, consisting of an initial extracellular alkalinization followed by a slower acidification. In vitro, stimulus trains also evoked biphasic pH shifts in the CA1 region. However, in CA1, seizure activity in vitro induced in the absence of synaptic transmission, by perfusing with 0 Ca(2+)/5 mM K(+) medium, was only associated with extracellular acidification. In the dentate gyrus in vivo, seizure activity induced by stimulation to the angular bundle or by injection of either bicuculline or kainic acid was only associated with extracellular acidification. In vitro, stimulus trains evoked only acidification. In the dentate gyrus in vitro, recurrent epileptiform activity induced in the absence of synaptic transmission by perfusion with 0 Ca(2+)/8 mM K(+) medium was associated with extracellular acidification. To test whether glial cell depolarization plays a role in the regulation of the extracellular pH, slices were perfused with 1 mM barium. Barium increased the amplitude of the initial alkalinization in CA1 and caused the appearance of alkalinization in the dentate gyrus. In both CA1 and the dentate gyrus in vitro, spreading depression was associated with biphasic pH shifts. These results demonstrate that activity-dependent extracellular pH shifts differ between CA1 and dentate gyrus both in vivo and in vitro. The differences in pH fluctuations with neuronal activity might be a marker for the basis of the regional differences in seizure susceptibility between CA1 and the dentate gyrus.

Sodium pump activity, not glial spatial buffering, clears potassium after epileptiform activity induced in the dentate gyrus.

A number of mechanisms have been proposed to play a role in the regulation of activity-dependent variations in extracellular potassium concentration ([K(+)](o)). We tested possible regulatory mechanisms for [K(+)](o) during spontaneous recurrent epileptiform activity induced in the dentate gyrus of hippocampal slices from adult rats by perfusion with 8 mM potassium and 0-added calcium medium in an interface chamber. Local application of tetrodotoxin blocked local [K(+)](o) changes, suggesting that potassium is released and taken up locally. Perfusion with barium or cesium, blockers of the inward rectifying potassium channel, did not alter the baseline [K(+)](o), the ceiling level of [K(+)](o) reached during the burst, or the rate of [K(+)](o) recovery after termination of the bursts. Decreasing gap junctional conductance did not change the baseline [K(+)](o) or the half-time of recovery of the [K(+)](o) after the bursts but did cause a decrease in the ceiling level of [K(+)](o). Perfusion with furosemide, which will block cation/chloride cotransporters, or perfusion with low chloride did not change the baseline [K(+)](o) or the half-time of recovery of the [K(+)](o) after the bursts but did increase the ceiling level of [K(+)](o). Bath or local application of ouabain, a Na(+)/K(+)-ATPase inhibitor, increased the baseline [K(+)](o), slowed the rate of [K(+)](o) recovery, and induced spreading depression. These findings suggest that potassium redistribution by glia only plays a minor role in the regulation of [K(+)](o) in this model. The major regulator of [K(+)](o) in this model appears to be uptake via a Na(+)/K(+)-ATPase, most likely neuronal.

Activity-dependent intracellular acidification correlates with the duration of seizure activity.

Synchronized neuronal activity (seizures) can appear in the presence or absence of synaptic transmission. Mechanisms of seizure initiation in each of these conditions have been studied, but relatively few studies have addressed seizure termination. In particular, how are seizures terminated in the absence of synaptic activity where there is no loss of excitatory drive or augmentation of inhibitory inputs? We have studied dynamic activity-dependent changes of intracellular pH in the absence of synaptic transmission using the fluorescent pH indicator carboxylseminaphthorhodafluo-1. During epileptiform activity we observed intracellular acidification, whereas between seizures the intracellular pH recovered. Experimental conditions that shortened the epileptiform discharge correlated with more rapid intracellular acidification. On the other hand, experimental manipulation of intracellular pH altered the duration of the seizure discharge, with acidification resulting in early termination of the epileptiform activity. These data show a direct relationship between the level of intracellular acidification and the duration of the seizures, suggesting that an intracellular pH-dependent process can terminate nonsynaptic neuronal synchronization.

Cesium induces spontaneous epileptiform activity without changing extracellular potassium regulation in rat hippocampus.

Cesium has been widely used to study the roles of the hyperpolarization-activated (I(h)) and inwardly rectifying potassium (K(IR)) channels in many neuronal and nonneuronal cell types. Recently, extracellular application of cesium has been shown to produce epileptiform activity in brain slices, but the mechanisms for this are not known. It has been proposed that cesium blocks the K(IR) in glia, resulting in an abnormal accumulation of potassium in the extracellular space and inducing epileptiform activity. This hypothesis has been tested in hippocampal slices and cultured hippocampal neurons using potassium-sensitive microelectrodes. In the present study, application of cesium produced spontaneous epileptiform discharges at physiological extracellular potassium concentration ([K(+)](o)) in the CA1 and CA3 regions of hippocampal slices. This epileptiform activity was not mimicked by increasing the [K(+)](o). The epileptiform discharges induced by cesium were not blocked by the N-methyl-D- aspartate (NMDA) receptor antagonist AP-5, but were blocked by the non-NMDA receptor antagonist CNQX. In the dentate gyrus, cesium induced the appearance of spontaneous nonsynaptic field bursts in 0 added calcium and 3 mM potassium. Moreover, cesium increased the frequency of field bursts already present. In contrast, ZD-7288, a specific I(h) blocker, did not cause spontaneous epileptiform activity in CA1 and CA3, nor did it affect the field bursts in the dentate gyrus, suggesting that cesium induced epileptiform activity is not directly related to blockade of the I(h). When potassium-sensitive microelectrodes were used to measure [K(+)](o), there was no significant increase in [K(+)](o) in CA1 and CA3 after cesium application. In the dentate gyrus, cesium did not change the baseline level of [K(+)](o) or the rate of [K(+)](o) clearance after the field bursts. In cultured hippocampal neurons, which have a large and relatively unrestricted extracellular space, cesium also produced cellular burst activity without significantly changing the resting membrane potential, which might indicate an increase in [K(+)](o). Our results suggest that cesium causes epileptiform activity by a mechanism unrelated to an alteration in [K(+)](o) regulation.

Astrocytic regulation of the recovery of extracellular potassium after seizures in vivo.

Glial cells are believed to play a major role in the regulation of the extracellular potassium concentration ([K+]o), particularly when the [K+]o is increased. Using ion-selective electrodes, we compared the [K+]o changes in the dentate gyrus of urethane-anaesthetized adult rats in the presence of reactive astrocytes and after reduction of glial function. The regulation of [K+]o in the dentate gyrus was determined by measuring the ceiling level of [K+]o and the half-time of recovery of [K+]o during and after seizures produced by 20 Hz trains of stimulation to the angular bundle. Reactive astrocytes were induced by repeated seizures and their presence was confirmed by a qualitative increase in glial fibrillary acidic protein (GFAP) and vimentin immunoreactivity. To inhibit glial function, fluorocitrate (FC), a reversible metabolic inhibitor, or alpha-aminoadipate (alpha-AA), an irreversible toxin, was injected into the dentate gyrus region, and the regulation of [K+]o was monitored for 8 h or 2 days later, respectively. After alpha-aminoadipate, loss of astrocytes in the dentate gyrus was demonstrated by loss of staining for GFAP. In the presence of reactive astrocytes there was no significant change in the peak [K+]o during seizures or the half-time of recovery of [K+]o after seizures compared to control animals. alpha-Aminoadipate significantly slowed the rate of recovery of [K+]o, but did not change the ceiling [K+]o. Fluorocitrate reversibly decreased the ceiling [K+]o, but also slowed the rate of recovery of [K+]o. Overall our results suggest that normal glial function is required for the recovery of elevated [K+]o after seizures in vivo.

Effects of huperzine A on nucleus basalis magnocellularis lesion-induced spatial working memory deficit.

AIM: To study the effects of huperzine A on nucleus basalis magnocellularis (NBM) lesion-induced spatial working memory impairment. METHODS: A delayed-non-match-to-sample radial arm maze task was used to study spatial working memory. The choline acetyltransferase (ChAT) activity was determined by the conversion of [3H]acetyl-CoA to [3H]ACh. RESULTS: Unilateral NBM lesion by kainic acid 0.02 mumol impaired rat's ability to perform this working memory task as evidenced by fewer correct choices after different delay intervals and more total errors to complete the task. This behavioral impairment associated with a decrease in the activity of ChAT by about 40% in the ipsilateral cerebral cortex. Huperzine A (0.2 mg.kg-1 i.p. 30 min before testing) ameliorated this spatial working memory impairment. Physostigmine (0.2-0.3 mg.kg-1 i.p. 20 min before testing) also attenuated the NBM lesion-induced memory deficit. CONCLUSION: The integrity of NBM is critical for spatial working memory processing, and this working memory impairment induced by NBM lesion can be ameliorated by huperzine A and physostigmine.

Effects of felbamate, gabapentin and lamotrigine on seizure parameters and excitability in the rat hippocampus.

The effects of three new antiepileptic drugs (felbamate, lamotrigine and gabapentin) on the parameters of seizure initiation and termination in one model of reverberatory seizures in the hippocampal-parahippocampal loop in urethane-anesthetized rats were determined. All three of the drugs caused a dose-dependent decrease in the duration of the seizure discharge. Only very high doses of felbamate had a significant effect on seizure initiation. In addition to the seizure model, we tested the three new anti-epileptic drugs for an effect on excitability, on paired-pulse inhibition and on long-term potentiation (LTP) in the dentate gyrus of the urethane-anesthetized rat. None of the drugs altered LTP or excitability. However, gabapentin altered paired-pulse inhibition, causing a loss of inhibition. For felbamate and lamotrigine, the effects of seizure duration are consistent with their clinical effects as antiepileptic drugs. However, the effect of gabapentin on paired-pulse inhibition suggests a proconvulsant effect and the effect on seizure duration suggests an antiepileptic effect.

Effect of N omega-nitro-L-arginine on working memory of rats in eight-arm maze task.

AIM: To study the effect of N omega-nitro-L-arginine (NNA) (a NO synthase inhibitor) on memory in the rats. METHODS: A delayed non-match-to-sample (DNMTS) eight-arm maze task was used to study spatial working memory. RESULTS: Intraperitoneal injection of NNA 100 mg kg-1 before the trial or at the start of the delay did not affect the accuracy, while pretraining administration of scopolamine 0.25 mg kg-1 produced impairment in mnemonic performance as evidenced by fewer correct choices after the delay and more total errors to complete the task. Intracerebroventricular infusion of NNA (10, 50, 100 nmol) did not affect the accuracy. CONCLUSION: A single ip or icv injection of NNA is not sufficient to alter the memory formation and use in this DNMTS eight-arm maze task.

Effect of huperzine A, a novel acetylcholinesterase inhibitor, on radial maze performance in rats.

Rats were trained to run in a spatial, radial arm maze using a procedure to determine two memory functions, working and reference memory. The muscarinic antagonist, not the nicotinic antagonist, impaired both working and reference memory of rats. Scopolamine (0.125, 0.15, and 0.2 mg/kg, IP, 30 min before a session) significantly impaired choice accuracy in the eight-arm maze. In contrast, mecamylamine (5, 10, and 15 mg/kg) did not affect the performance. Huperzine A (0.1, 0.2, and 0.3 mg/kg, IP, 30 min before testing) and physostigmine (0.3 mg/kg, IP, 20 min before testing) could reverse scopolamine-induced deficits in the task. Chronic treatment with huperzine A (0.25 mg/kg, PO, once a day) for 8 consecutive days was as potent as acute treatment on attenuating the scopolamine-induced amnesia.

Effects of isovanihuperzine A on cholinesterase and scopolamine-induced memory impairment.

AIM: To study the effects of isovanihuperzine A (IVHA) on cholinesterase and scopolamine-induced memory deficit. METHODS: AChE and BuChE activities were determined by the colorimetric method of Ellman. The Ki value was determined by the plotting method of Lineweaver and Burk. In a behavioral test, rats were trained to perform a radial arm maze task using a partially baited procedure. RESULTS: The anti-AChE activity of IVHA was comparable to huperzine A (Hup-A), and was more potent than those of physostigmine and galanthamine with an IC50 value of 0.11 mumol.L-1. IVHA was a mixed competitive type with a Ki value of 32 nmol.L-1. It bound to AChE in a reversible manner. IVHA at a dose of 0.2 mg.kg-1 ip significantly reversed scopolamine-induced working memory and reference memory impairments in radial arm maze. CONCLUSION: IVHA is a new potential reversible AChE inhibitor and merits further study as a cognitive enhancer.

[Metastatic cancer of the endocardium].

16 cases of metastatic cancer in the endocardium collected from 2,050 autopsy cases in Baptist Medical Center, Missouri, USA from 1961 to 1983 are reported. There were 484 malignant tumors and 68 metastatic cancers in the heart, in which 16 involved the endocardium comprising 0.8% of the total autopsies during the same period, 3.3% of the malignant tumors and 23.5% of the metastatic cancer in the heart. Primary sites of these 16 cases are as follows: malignant melanoma 6 cases, lung cancer 2, thyroid carcinoma 2, rectal cancer 2, breast, liver, kidney and skin 1 case each. Metastatic cancer in the endocardium is rare. Diagnosis is commonly made by occurrent findings during autopsy. The primary lesions, metastatic sites and pathological and clinical features are discussed.

[Metastatic cancer of the heart--a clinicopathologic study of 68 cases].

The study of 68 autopsy cases of metastatic cancer of the heart is reported. There were 41 male and 27 female. The age was from 26-89 years. The primary neoplasms are as follows: carcinoma of lung (26.5%), lymphosarcoma (14.7%), leukemia (11.8%), malignant melanoma (8.8%), carcinoma of breast (7.4%) and of kidney (7.4%). The laterality ratio of the heart was; right: left is 1:2. The myocardium was involved in 50 cases, the pericardium, 30 cases and the endocardium, 16 cases. 19 cases (27.9%) had symptoms referable to the metastatic disease in the heart. None of the patient was correctly diagnosed before death as to the metastatic lesion in the heart. 88.2% (60/68) of cases died of general metastases, in which 14 succumbed directly from metastatic cancer of the heart.