The right dorsolateral prefrontal cortex is essential in seconds range timing, but not in milliseconds range timing: An investigation with transcranial direct current stimulation.

It is unclear whether altering the activity of the right dorsolateral prefrontal cortex (right DLPFC) affects an individual's timing performance in milliseconds- and seconds range timing. Here we investigated the causal role of right DLPFC in milliseconds- and seconds range timing with a temporal bisection task under the application of transcranial direct current stimulation (tDCS) that altered the neural activities of the right DLPFC. The tDCS conditions consisted of anodal, cathodal, and sham conditions. The electrodes were placed over the F4 position (10-20 system) and on the left supraorbital forehead. In current study, participants completed two blocks of trials involving short ("Short blocks": 200-800 ms) or longer ("Long blocks": 1400-2600 ms) durations. The results showed that no significant differences in the bisection point (BP) were found among anodal condition, sham condition and cathodal condition in "Short blocks". However, in "Long blocks", the BP were found to be shifted toward the left for the anodal condition, sham condition, compared to cathodal condition, suggesting that the stimulus duration was judged to last longer for anodal condition compared to sham condition, whereas shorter for cathodal condition compared to sham condition. The results demonstrated that the right DLPFC played a causal role in seconds range timing (average duration 2000 ms), but not in milliseconds range timing (average duration 500 ms), which is shown it might be involved in the cognitive processing (for example, working memory process) based on dual-timing system and scalar timing theory.

Inflammation-induced GluA1 trafficking and membrane insertion of Ca2+ permeable AMPA receptors in dorsal horn neurons is dependent on spinal tumor necrosis factor, PI3 kinase and protein kinase A.

Peripheral inflammation induces sensitization of nociceptive spinal cord neurons. Both spinal tumor necrosis factor (TNF) and neuronal membrane insertion of Ca2+ permeable AMPA receptor (AMPAr) contribute to spinal sensitization and resultant pain behavior, molecular mechanisms connecting these two events have not been studied in detail. Intrathecal (i.t.) injection of TNF-blockers attenuated paw carrageenan-induced mechanical and thermal hypersensitivity. Levels of GluA1 and GluA4 from dorsal spinal membrane fractions increased in carrageenan-injected rats compared to controls. In the same tissue, GluA2 levels were not altered. Inflammation-induced increases in membrane GluA1 were prevented by i.t. pre-treatment with antagonists to TNF, PI3K, PKA and NMDA. Interestingly, administration of TNF or PI3K inhibitors followed by carrageenan caused a marked reduction in plasma membrane GluA2 levels, despite the fact that membrane GluA2 levels were stable following inhibitor administration in the absence of carrageenan. TNF pre-incubation induced increased numbers of Co2+ labeled dorsal horn neurons, indicating more neurons with Ca2+ permeable AMPAr. In parallel to Western blot results, this increase was blocked by antagonism of PI3K and PKA. In addition, spinal slices from GluA1 transgenic mice, which had a single alanine replacement at GluA1 ser 845 or ser 831 that prevented phosphorylation, were resistant to TNF-induced increases in Co2+ labeling. However, behavioral responses following intraplantar carrageenan and formalin in the mutant mice were no different from littermate controls, suggesting a more complex regulation of nociception. Co-localization of GluA1, GluA2 and GluA4 with synaptophysin on identified spinoparabrachial neurons and their relative ratios were used to assess inflammation-induced trafficking of AMPAr to synapses. Inflammation induced an increase in synaptic GluA1, but not GluA2. Although total GluA4 also increased with inflammation, co-localization of GluA4 with synaptophysin, fell short of significance. Taken together these data suggest that peripheral inflammation induces a PI3K and PKA dependent TNFR1 activated pathway that culminates with trafficking of calcium permeable AMPAr into synapses of nociceptive dorsal horn projection neurons.

Slow development of ALS-like spinal cord pathology in mutant valosin-containing protein gene knock-in mice.

Pathological features of amyotrophic lateral sclerosis (ALS) include, in addition to selective motor neuron (MN) degeneration, the occurrence of protein aggregates, mitochondrial dysfunction and astrogliosis. SOD1 mutations cause rare familial forms of ALS and have provided the most widely studied animal models. Relatively recent studies implicating another protein, TDP-43, in familial and sporadic forms of ALS have led to the development of new animal models. More recently, mutations in the valosin-containing protein (VCP) gene linked to the human genetic disease, Inclusion Body Myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD), were found also to be associated with ALS in some patients. A heterozygous knock-in VCP mouse model of IBMPFD (VCP(R155H/+)) exhibited muscle, bone and brain pathology characteristic of the human disease. We have undertaken studies of spinal cord pathology in VCP(R155H/+) mice and find age-dependent degeneration of ventral horn MNs, TDP-43-positive cytosolic inclusions, mitochondrial aggregation and progressive astrogliosis. Aged animals (~24-27 months) show electromyography evidence of denervation consistent with the observed MN loss. Although these animals do not develop rapidly progressive fatal ALS-like disease during their lifespans, they recapitulate key pathological features of both human disease and other animal models of ALS, and may provide a valuable new model for studying events preceding onset of catastrophic disease.

Invasive pulmonary aspergillosis in patients with HBV-related liver failure.

Invasive pulmonary aspergillosis (IPA) has been increasingly frequent in severe liver disease. We aim to investigate the clinical presentation, predisposing factors, and treatment of IPA in patients with liver failure caused by hepatitis B virus (HBV) infection. Medical records from 798 patients with HBV-related liver failure were reviewed. A total of 43 patients with probable IPA were selected as the case group, another 43 patients with bacterial infection and 43 patients without any infections were selected, for whose age, sex, date of admission, and the disease onset were matched with the case group. We evaluated the risk factors, clinical manifestations, treatment, and subsequent outcome of IPA in patients with HBV-related liver failure. Multivariate logistic regression models were used to demonstrate risk factors associated with IPA. Compared with patients with bacterial infection and those without any infection, patients with probable IPA used more antibiotics and steroids, and had poorer conditions and the highest mortality (P < 0.0001). Multiple antibiotics use and frequent invasive procedures were independent factors associated with the occurrence of IPA in patients with HBV-related liver failure. Patients with HBV-related liver failure are predisposed to IPA and may have a more severe condition and poorer prognosis.

AMPA/kainate receptor-triggered Zn2+ entry into cortical neurons induces mitochondrial Zn2+ uptake and persistent mitochondrial dysfunction.

Rapid Zn2+ influx through Ca2+-permeable AMPA/kainate (Ca-A/K) channels triggers reactive oxygen species (ROS) generation and is potently neurotoxic. The first aim of this study was to determine whether these effects might result from direct mitochondrial Zn2+ uptake. Adapting the mitochondrially sequestered divalent cation sensitive probe, rhod-2, to visualize mitochondrial Zn2+, present studies indicate that Zn2+ is taken up into these organelles. The specificity of the signal for Zn2+ was indicated by its reversal by Zn2+ chelation, and its mitochondrial origin indicated by its speckled extranuclear appearance and by its elimination upon pretreatment with the mitochondrial protonophore, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). Consistent with inhibition of mitochondrial Zn2+ uptake, FCCP also slowed the recovery of cytosolic Zn2+ elevations in Ca-A/K(+) neurons. Further studies sought clues to the high toxic potency of intracellular Zn2+. In experiments using the mitochondrial membrane polarization (DeltaPsi(m))-sensitive probe tetramethylrhodamine ethyl ester and the ROS-sensitive probe hydroethidine, brief kainate exposures in the presence of 300 microM Zn2+ (with or without Ca2+) resulted in prolonged loss of DeltaPsi(m) and corresponding prolonged ROS generation in Ca-A/K(+) neurons, in comparison to the more rapid recovery from loss of DeltaPsi(m) and transient ROS generation after kainate/1.8 mM Ca2+ exposures.

AMPA exposures induce mitochondrial Ca(2+) overload and ROS generation in spinal motor neurons in vitro.

The reason for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is primarily unknown. A possible factor is the expression by motor neurons of Ca(2+)-permeable AMPA/kainate channels, which may permit rapid Ca(2+) influx in response to synaptic receptor activation. However, other subpopulations of central neurons, most notably forebrain GABAergic interneurons, consistently express large numbers of these channels but do not degenerate in ALS. Indeed, when subjected to identical excitotoxic exposures, motor neurons were more susceptible than GABAergic neurons to AMPA/kainate receptor-mediated neurotoxicity. Microfluorimetric studies were performed to examine the basis for the difference in vulnerability. First, AMPA or kainate exposures appeared to trigger substantial mitochondrial Ca(2+) loading in motor neurons, as indicated by a sharp increase in intracellular Ca(2+) after addition of the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl hydrazone (FCCP) after the agonist exposure. The same exposures caused little mitochondrial Ca(2+) accumulation in GABAergic cortical neurons. Subsequent experiments examined other measures of mitochondrial function to compare sequelae of AMPA/kainate receptor activation between these populations. Brief exposure to either AMPA or kainate caused mitochondrial depolarization, assessed using tetramethylrhodamine ethylester, and reactive oxygen species (ROS) generation, assessed using hydroethidine, in motor neurons. However, these effects were only seen in the GABAergic neurons after exposure to the nondesensitizing AMPA receptor agonist kainate. Finally, addition of either antioxidants or toxins (FCCP or CN(-)) that block mitochondrial Ca(2+) uptake attenuated AMPA/kainate receptor-mediated motor neuron injury, suggesting that the mitochondrial Ca(2+) uptake and consequent ROS generation are central to the injury process.

Glutamate triggers preferential Zn2+ flux through Ca2+ permeable AMPA channels and consequent ROS production.

ZN2+ co-released with glutamate at excitatory synaptic sites can enter and cause injury to postsynaptic neurons. While prior studies using the slowly desensitizing agonist kainate suggested preferential Zn2+ permeation through Ca2+ permeable AMPA/kainate (Ca-A/K) channels, the present study aims to assess relevance of those findings upon more physiological receptor activation. Microfluorimetric techniques were used to measure [Zn2+]i attained upon exposure to the rapidly desensitizing agonist AMPA or to the physiological agonist glutamate, in the presence of 300 microM Zn2+. Under these conditions, micromolar [Zn2+]i rises (delta[Zn2+]i) were still observed to occur selectively in the subset of neurons that express large numbers of Ca-A/ K channels. Further studies using the oxidation sensitive dye, hydroethidine, revealed Zn2+-dependent reactive oxygen species generation that paralleled delta[Zn2+]i, with rapid oxidation only observed in the case of Zn2+ entry through Ca-A/K channels.

Dendritic localization of Ca(2+)-permeable AMPA/kainate channels in hippocampal pyramidal neurons.

Although it is well established that cortical and hippocampal gamma-aminobutyric acid (GABA)-ergic neurons generally have large numbers of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate channels (Ca-A/K channels), their presence on pyramidal neurons is controversial. Ca2+ permeability of AMPA channels is regulated by expression of a particular glutamate receptor subunit (GluR2), which confers Ca2+ impermeability to heteromeric channels. Most electrophysiology studies, as well as in situ hybridization and immunolabeling studies demonstrating expression of GluR2 mRNA or peptide in pyramidal neurons, have provided evidence against the presence of Ca-A/K channels on pyramidal neurons. However, observations that pyramidal neurons often appear to be labeled by kainate-stimulated Co2+ influx (Co2+(+) cells), a histochemical stain that identifies cells possessing Ca-A/K channels, suggests that they may have these channels. The present study futher examines cellular and subcellular distribution of Ca-A/K channels on hippocampal pyramidal neurons in slice as well as in culture. To this end, techniques of kainate-stimulated Co2+ influx labeling, supplemented by AMPA receptor subunit immunocytochemistry and fluorescent imaging of kainate-stimulated intracellular Ca2+ ([Ca2+]i) rises are employed. Co2+ labeling is often seen in pyramidal neuronal dendrites in both slice and in culture. In addition, although GluR1 and 4 staining in these neurons is often seen in the soma and dendrites, GluR2 label, when evident, is generally more restricted to the soma. Finally, measurement of kainate-stimulated [Ca2+]i rises in cultured neurons, assessed by using low affinity Ca2+ indicators in the presence of N-methyl-D-aspartate (NMDA) receptor and voltage-sensitive Ca2+ channel blockade, often shows dendritic rises to precede those in the somata. Thus, these data support the hypothesis that Ca-A/K channels are present in dendritic domains of many pyramidal neurons, and may help to provide resolution of the apparently conflicting data regarding their distribution.

Prostate specific antigen is detectable in formalin-fixed semen.

OBJECTIVE: To conduct a pilot study to test whether or not prostate specific antigen (PSA) and/or PSA-positive cells can be detected and characterized in semen specimens archived in aldehyde fixative. MATERIALS AND METHODS: Specimens from 12 men were examined, six before elective vasectomy and six undergoing infertility evaluation. Fixed semen elements were assessed immunocytologically using monoclonal antibodies against PSA and leucocyte common antigen, CD 45, as a control. RESULTS: PSA was detectable in the semen from the fertile men as amorphous protein and contained within round vesicles (prostasomes). Semen from men undergoing infertility evaluation contained a greater variation in detectable forms and amount of PSA than the specimens from the fertile men, including PSA associated with some nonspermatozoal cells (NSCs). CONCLUSION: PSA is detectable by immunocytological analysis of semen specimens archived in aldehyde-based fixative. Three forms of PSA were detected; within round vesicles characteristic of prostasomes, associated with some NSCs, and as amorphous protein. The detected variations suggest that analysis of PSA-positive semen elements may provide important insights into prostate physiology.

Preferential Zn2+ influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production.

Synaptically released Zn2+ can enter and cause injury to postsynaptic neurons. Microfluorimetric studies using the Zn2+-sensitive probe, Newport green, examined levels of [Zn2+]i attained in cultured cortical neurons on exposure to N-methyl-D-asparte, kainate, or high K+ (to activate voltage-sensitive Ca2+ channels) in the presence of 300 microM Zn2+. Indicating particularly high permeability through Ca2+-permeable alpha-amino3-hydroxy-5-methyl-4-isoxazolepropionic-acid/kainate (Ca-A/K) channels, micromolar [Zn2+]i rises were observed only after kainate exposures and only in neurons expressing these channels [Ca-A/K(+) neurons]. Further studies using the oxidation-sensitive dye, hydroethidine, revealed Zn2+-dependent reactive oxygen species (ROS) generation that paralleled the [Zn2+]i rises, with rapid oxidation observed only in the case of Zn2+ entry through Ca-A/K channels. Indicating a mitochondrial source of this ROS generation, hydroethidine oxidation was inhibited by the mitochondrial electron transport blocker, rotenone. Additional evidence for a direct interaction between Zn2+ and mitochondria was provided by the observation that the Zn2+ entry through Ca-A/K channels triggered rapid mitochondrial depolarization, as assessed by using the potential-sensitive dye tetramethylrhodamine ethylester. Whereas Ca2+ influx through Ca-A/K channels also triggers ROS production, the [Zn2+]i rises and subsequent ROS production are of more prolonged duration.

Human sperm cryobanking. Use of modified liquid nitrogen vapor.

OBJECTIVE: To study a modified liquid nitrogen vapor cryopreservation technique for human spermatozoa. STUDY DESIGN: The freezing rate of the modified liquid nitrogen vapor method was controlled using thermocouple probes to simultaneously measure the temperature of both the liquid nitrogen vapor and semen specimen. Sperm cryosurvival and postthaw velocity were compared using a programmable biologic freezer and our modified vapor freezing procedure. RESULTS: There were no significant differences in postthaw sperm viability between the two methods. CONCLUSION: This modified vapor freezing technique permits controlled cooling that is efficient, rapid and inexpensive.

Rapid Ca2+ entry through Ca2+-permeable AMPA/Kainate channels triggers marked intracellular Ca2+ rises and consequent oxygen radical production.

The widespread neuronal injury that results after brief activation of highly Ca2+-permeable NMDA channels may, in large part, reflect mitochondrial Ca2+ overload and the consequent production of injurious oxygen radicals. In contrast, AMPA/kainate receptor activation generally causes slower toxicity, and most studies have not found evidence of comparable oxygen radical production. Subsets of central neurons, composed mainly of GABAergic inhibitory interneurons, express AMPA/kainate channels that are directly permeable to Ca2+ ions. Microfluorometric techniques were performed by using the oxidation-sensitive dye hydroethidine (HEt) to determine whether the relatively rapid Ca2+ flux through AMPA/kainate channels expressed on GABAergic neurons results in oxygen radical production comparable to that triggered by NMDA. Consistent with previous studies, NMDA exposures triggered increases in fluorescence in most cultured cortical neurons, whereas high K+ (50 mM) exposures (causing depolarization-induced Ca2+ influx through voltage-sensitive Ca2+ channels) caused little fluorescence change. In contrast, kainate exposure caused fluorescence increases in a distinct subpopulation of neurons; immunostaining for glutamate decarboxylase revealed the responding neurons to constitute mainly the GABAergic population. The effect of NMDA, kainate, and high K+ exposures on oxygen radical production paralleled the effect of these exposures on intracellular Ca2+ levels when they were monitored with the low-affinity Ca2+-sensitive dye fura-2FF, but not with the high-affinity dye fura-2. Inhibition of mitochondrial electron transport with CN- or rotenone almost completely blocked kainate-triggered oxygen radical production. Furthermore, antioxidants attenuated neuronal injury resulting from brief exposures of NMDA or kainate. Thus, as with NMDA receptor activation, rapid Ca2+ influx through Ca2+-permeable AMPA/kainate channels also may result in mitochondrial Ca2+ overload and consequent injurious oxygen radical production.

Kainate-stimulated Zn2+ uptake labels cortical neurons with Ca2+-permeable AMPA/kainate channels.

The endogenous cation, Zn2+, is synaptically released and may trigger neurodegeneration after permeating through NMDA channels, voltage sensitive Ca2+ channels (VSCC), or Ca2+ permeable AMPA/kainate channels (Ca-A/K). Neurons expressing Ca-A/K can be identified by a histochemical stain based upon kainate-stimulated Co2+ uptake (Co2+(+) neurons). The primary objective of this study was to determine whether a similar approach could be employed to visualize agonist-stimulated intracellular Zn2+ accumulation, and, thus, to test the hypothesis that Ca-A/K permit particularly rapid Zn2+ flux. Substituting Zn2+ for Co2+ during agonist-stimulated uptake, followed by Timm's sulfide-silver staining to visualize intracellular Zn2+, resulted in distinct labeling of a subpopulation of cortical neurons (Zn2+(+) neurons) closely resembling Co2+(+) neurons, suggesting that, like Co2+, Zn2+ may permeate Ca-A/K with particular rapidity. Neither NMDA nor high K+ triggered comparable Zn2+ accumulation, indicating substantially greater permeation through Ca-A/K than through NMDA channels or VSCC. Both fluorescence studies of intracellular Zn2+ accumulation and double staining studies (using SMI-32 and anti-glutamate decarboxylase antibodies, both markers of cortical neuronal subsets), support the contention that Zn2+ and Co2+ labeling identify a common set of neurons characterized by expression of AMPA/kainate channels directly permeable to Zn2+ and Co2+ as well as Ca2+. Furthermore, the preferential destruction of Zn2+(+) neurons (like Co2+(+) neurons) after brief kainate exposures in the presence of lower, more physiologic concentrations of Zn2+ suggests that Zn2+ permeation through Ca-A/K could contribute to selective neurodegeneration in disease. Finally, the study provides a novel and potentially advantageous histochemical approach for kainate-stimulated Co2+ or Zn2+ uptake labeling, using a room temperature technique (Timm's staining) rather than the usual hot AgNO3 development of the Co2+ stain.

Study of coordination equilibria in systems of coexisting mononuclear and polynuclear complexes by an isosbestic point-spectrophotometric method.

This paper reports a method for determining the stability constants of complexes in a system of coexisting mononuclear and polynuclear complexes based on analysis of dual isosbestic points. First it is necessary to determine the dissociation degrees of either of the two complexes concerned in two directions, and then their stability constants. With this method, the conditional constant can be evaluated by appropriate treatment and selection of the proper equations for solutions. For demonstration purposes the scandium/Xylenol Orange system was used to test the model and satisfactory results were obtained.

Ca(2+)-permeable AMPA/kainate and NMDA channels: high rate of Ca2+ influx underlies potent induction of injury.

Neurodegeneration may occur secondary to glutamate-triggered Ca2+ influx through any of three routes: NMDA channels, voltage-sensitive Ca2+ channels (VSCC), and Ca(2+)-permeable AMPA/kainate channels (Ca-A/K). This study aims to examine Ca2+ ion dynamics in the generation of excitotoxic injury by correlating the relative amounts of 45Ca2+ that flow into cortical neurons through each of these routes over a 10 min epoch ("10 min Ca2+ loads;" a measure of influx rate), with resultant levels of intracellular free Ca2+ ([Ca2+]) and subsequent injury. Neurons possessing Ca-A/K make up a small subset (approximately 13%) of cortical neurons in culture, which can be identified by a histochemical stain based on kainate-stimulated Co2+ uptake (Co2+ (+) neurons) and which are unusually vulnerable to AMPA/kainate receptor-mediated injury. Initial studies using brief kainate exposures (to selectively destroy Co2+ (+) neurons) along with kainate-triggered 45Ca2+ influx measurements suggested that kainate causes rapid Ca2+ influx into Co2+ (+) neurons (comparable to that caused by NMDA). Influx through both Ca-A/K and NMDA channels increased proportionately with extracellular Ca2+, suggesting that these channels have high Ca2+ permeability. When cultures were subjected to exposures that gave similar 10 min Ca2+ loads through different routes, comparable levels of injury were observed, suggesting that net intracellular Ca2+ accumulation is a critical determinant of injury. However, the relationship between [Ca2+]i and influx was less direct: although exposures that gave the lowest or highest 10 min Ca2+ loads showed correspondingly lower or higher mean [Ca2+]i responses, there appears to be a wide range of exposures over which individual neuronal differences and sequestration/buffering mechanisms obscure [Ca2+]i as a reflection of influx rate.

Motor neurons are selectively vulnerable to AMPA/kainate receptor-mediated injury in vitro.

The nonphosphorylated neurofilament marker SMI-32 stains motor neurons in spinal cord slices and stains a subset of cultured spinal neurons ["large SMI-32(+) neurons"], which have a morphology consistent with motor neurons identified in vitro: large cell body, long axon, and extensive dendritic arborization. They are found preferentially in ventral spinal cord cultures, providing further evidence that large SMI-32(+) neurons are indeed motor neurons, and SMI-32 staining often colocalizes with established motor neuron markers (including acetylcholine, calcitonin gene-related peptide, and peripherin). Additionally, choline acetyltransferase activity (a frequently used index of the motor neuron population) and peripherin(+) neurons share with large SMI-32(+) neurons an unusual vulnerability to AMPA/kainate receptor-mediated injury. Kainate-induced loss of these motor neuron markers is Ca2+-dependent, which supports a critical role of Ca2+ ions in this injury. Raising extracellular Ca2+ exacerbates injury, whereas removal of extracellular Ca2+ is protective. A basis for this vulnerability is provided by the observation that most peripherin(+) neurons, like large SMI-32(+) neurons, are subject to kainate-stimulated Co2+ uptake, a histochemical stain that identifies neurons possessing Ca2+-permeable AMPA/kainate receptor-gated channels. Finally, of possibly greater relevance to the slow motor neuronal degeneration in diseases, both large SMI-32(+) neurons and peripherin(+) neurons are selectively damaged by prolonged (24 hr) low-level exposures to kainate (10 microM) or to the glutamate reuptake blocker L-trans-pyrrolidine-2,4-dicarboxylic acid (100 microM). During these low-level kainate exposures, large SMI-32(+) neurons showed higher intracellular Ca2+ concentrations than most spinal neurons, suggesting that Ca2+ ions are also important in this more slowly evolving injury.

Zn(2+) permeates Ca(2+) permeable AMPA/kainate channels and triggers selective neural injury.

Brief exposures of cortical cultures to kainate (100 mu M) plus Zn(2+) (300 mu M) cause fluorescence of the Zn(2+) sensitive dye, TS-Q, to appear in virtually all neurons, probably reflecting depolarization and secondary Zn(2+) permeation through voltage-sensitive Ca(2+) channels. However, if Na+ ions are removed from the media (to prevent depolarization), prominent TS-Q fluorescence is still observed in the small subset of neurons labeled by kainate stimulated Co(2+) uptake (Co(2+)(+) neurons), a histochemical technique that identifies neurons expressing Ca(2+) permeable AMPA/kainate receptor-gated channels. Kainate/Zn(2+) exposures in Na+ containing media with lower (50-100 mu M) Zn(2+) concentrations resulted 24 h later in selective loss of the Co(2+)(+) neurons, suggesting that these channels may permit particularly high rates of Zn(2+) passage. Thus, direct permeation of synaptically released Zn(2+) through Ca(2+) permeable AMPA/kainate channels could contribute to selective degeneration of neurons in disease as well as subserving physiological signaling functions.

Spinal cord neurons are vulnerable to rapidly triggered kainate neurotoxicity in vitro.

Initial studies found glutamate injury to murine spinal cultures (14-17 days in vitro) to reflect contributions of both NMDA and AMPA/kainate receptors. Subsequent experiments found the spinal cultures to be more sensitive than cortical cultures to injury from prolonged low level kainate exposures, and, unlike cortical cultures, to be significantly damaged by relatively brief (30-60 min) kainate exposures. This rapidly triggered kainate damage to spinal neurons is Ca(2+)-dependent. Also, more than 40% of spinal neurons (in comparison to about 15% of cortical neurons) are subject to kainate-activated Co2+ uptake (Co2+(+) neurons), a histochemical technique that labels neurons with Ca(2+)-permeable AMPA/kainate channels. These spinal Co2+(+) neurons are very sensitive to Ca(2+)-dependent kainate injury, and show greater kainate-induced elevations in intracellular Ca2+ concentrations ([Ca2+]i) than other spinal neurons during low level kainate exposures. Thus, the heightened vulnerability of spinal neurons to kainate toxicity may at least in part reflect the large proportion that possess Ca2+ permeable AMPA/kainate channels, permitting receptor activation to trigger rapid Ca2+ influx and overwhelm the cells Ca2+ homeostatic capabilities.

Semen leukocytes: friends or foes?

OBJECTIVE: To test the hypothesis that male reproductive tract leukocytes function in the elimination of abnormal spermatozoa from ejaculated semen. DESIGN: Semen specimens with > or = 2 x 10(6) nonspermatozoal cells/mL were examined for leukocytes and for mature sperm with ideal morphology. SETTING: Andrology laboratory of a Center of Assisted Reproductive Technology. RESULTS: Semen specimens with elevated concentrations of leukocytes contained a significantly higher frequency of sperm with ideal morphology than semen specimens with elevated numbers of immature germ cells and low numbers of leukocytes. CONCLUSIONS: The direct correlation between leukocyte density and sperm with ideal morphology supports the concept that sperm surveillance is a normal function of male reproductive tract leukocytes. Understanding such germ cell-leukocyte interactions may provide valuable new insights into immunologic control mechanisms in male reproductive tract tissues.

Ca2+ permeable AMPA/kainate channels permit rapid injurious Ca2+ entry.

Small subsets of central neurons possessing Ca2+ permeable AMPA/kainate channels can be identified by a histochemical stain based on kainate-stimulated Co2+ uptake (Co2+(+)neurons) and are unusually vulnerable to AMPA/kainate receptor-mediated injury. Using brief kainate exposures (which selectively destroy Co2+(+) neurons) along with kainate triggered 45Ca2+ influx measurements, we estimate kainate to cause an unusually high rate of Ca2+ influx into Co2+(+) neurons. Also, while fura-2 Ca2+ imaging revealed low (10 microM) kainate exposures to preferentially induce intracellular free Ca2+ ([Ca2+]i) elevations in Co2+(+) neurons, intense (100 microM) kainate exposures used in the 45Ca2+ influx studies triggered comparable [Ca2+]i rises in all neurons. These findings suggest that the exceptional vulnerability of Co2+(+) neurons to AMPA/kainate receptor-mediated injury reflects a high rate of agonist triggered Ca2+ influx, and that [Ca2+]i rises may only poorly reflect influx rate.