Behavioural responses of stable flies to cattle manure slurry associated odourants.

Stable flies (Stomoxys calcitrans [Diptera: Muscidae] L.) are blood-feeding synanthropic pests, which cause significant economic losses in livestock. Stable fly antennae contain olfactory sensilla responsive to host and host environment-associated odours. Field observation indicated that the abundance of stable flies increased significantly in grasslands or crop fields when cattle manure slurry was applied. Major volatile compounds emanating from manure slurry were collected and identified. Behavioural responses of stable flies to those compounds were investigated in laboratory bioassays and field-trapping studies. Results from olfactometer assays revealed that phenol, p-cresol and m-cresol were attractive to adult stable flies. When tested individually, attraction was higher with lower dosages. Stable flies were most attracted to blends of phenol and m-cresol or p-cresol. Traps with binary blend lures caught more stable flies in field trials as well.

Chronic network stimulation enhances evoked action potentials.

Neurons cultured on multielectrode arrays almost always lack external stimulation except during the acute experimental phase. We have investigated the effects of chronic stimulation during the course of development in cultured hippocampal neural networks by applying paired pulses at half of the electrodes for 0, 1 or 3 r/day for 8 days. Spike latencies increased from 4 to 16 ms as the distance from the stimulus increased from 200 to 1700 microm, suggesting an average of four synapses over this distance. Compared to no chronic stimulation, our results indicate that chronic stimulation increased evoked spike counts per stimulus by 50% at recording sites near the stimulating electrode and increased the instantaneous firing rate. On trials where both pulses elicited responses, spike count was 40-80% higher than when only one of the pulses elicited a response. In attempts to identify spike amplitude plasticity, we found mainly amplitude variation with different latencies suggesting recordings from neurons with different identities. These data suggest plastic network changes induced by chronic stimulation that enhance the reliability of information transmission and the efficiency of multisynaptic network communication.

Neuron network activity scales exponentially with synapse density.

Neuronal network output in the cortex as a function of synapse density during development has not been explicitly determined. Synaptic scaling in cortical brain networks seems to alter excitatory and inhibitory synaptic inputs to produce a representative rate of synaptic output. Here, we cultured rat hippocampal neurons over a three-week period to correlate synapse density with the increase in spontaneous spiking activity. We followed the network development as synapse formation and spike rate in two serum-free media optimized for either (a) neuron survival (Neurobasal/B27) or (b) spike rate (NbActiv4). We found that while synaptophysin synapse density increased linearly with development, spike rates increased exponentially in developing neuronal networks. Synaptic receptor components NR1, GluR1 and GABA-A also increase linearly but with more excitatory receptors than inhibitory. These results suggest that the brain's information processing capability gains more from increasing connectivity of the processing units than increasing processing units, much as Internet information flow increases much faster than the linear number of nodes and connections.

Pertussis toxin B-oligomer suppresses human immunodeficiency virus-1 Tat-induced neuronal apoptosis through feedback inhibition of phospholipase C-beta by protein kinase C.

Human immunodeficiency virus (HIV)-1 Tat is a multifunctional protein involved in viral replication, inflammation and apoptosis. Tat activates phospholipase C-beta (PLC-beta), presumably via a pertussis toxin (PTX) sensitive G(i) protein, which is critical for neuronal apoptosis. In this study, we show that Tat-mediated intracellular Ca(2+) release in rat pheochromocytoma (PC-12) cells and rat primary cortical neuronal cultures was abrogated by pretreatment with either pertussis toxin and/or its B-oligomer subunit (PTX-B), devoid of ADP ribosyltransferase activity. PTX-B pretreatment also inhibited intracellular Ca(2+) release by bradykinin and 2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl) benzenesulfonamide (m-3M3FBS), a director activator of phospholipase C. Activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PdBu) mimicked the PTX-B-mediated inhibition of m-3M3FBS-stimulated intracellular Ca(2+) increase, while inhibition of PKC by bisindolylmaleimide I hydrochloride (BIM) reversed the inhibitory action of PTX-B. Functionally, PTX-B reduced Tat-induced Bax and caspase-3 proteins and reduced cell apoptosis. We conclude that PTX inhibition of Tat-mediated intracellular Ca(2+) release is independent of ADP ribosylation of the G(i) protein via the A protomer, but mediated by the B-oligomer. Furthermore, PTX-B suppresses HIV-1 Tat-mediated apoptosis by reducing its activation of PLC-beta through a PKC activation pathway.

Dopamine and Abeta-induced stress signaling and decrements in Ca2+ buffering in primary neonatal hippocampal cells are antagonized by blueberry extract.

We have shown previously that dietary blueberry (BB) extract supplementation (S) reversed several parameters of neuronal and behavioral (e.g., cognition) aging in rodents. Additionally, findings indicate that COS-7 cells transfected with muscarinic receptor subtypes (e.g., M1) showed decrements in Ca;{2+} clearance following depolarization (Ca;{2+} Recovery time, Ca;{2+}RT) that were antagonized by BB. Since it has been postulated that at least part of the loss of cognitive function in aging may be dependent upon a dysregulation in calcium homeostasis (i.e., Ca;{2+}RT), we assessed whether: a) Ca;{2+}RT would be altered in dopamine (DA)- or amyloid beta (Abeta)-exposed cultured primary hippocampal neuronal cells (HNC), and b) BB pre-treatment of the cells would prevent these deficits. Thus, control or BB (0.5 mg/ml)-treated HNC were exposed to DA (0.1 mM, 2 hrs), Abeta(40) (25 microM, 24 hrs), Abeta(42) (25 microM, 24 hrs), and Abeta(25-35) (25 microM, 24 hrs), and Ca;{2+}RT following KCl-induced depolarization assessed. Ca;{2+}RT was assessed as the % of HNC showing recovery to 50%-70% of control at 5, 10, or 15 min after depolarization. Results indicated that DA significantly lowered Ca;{2+}RT in the HNC at all time points examined after depolarization. However, BB treatment selectively prevented these declines in Ca;{2+}RT. In the case of Abeta, the greatest effects on Ca;{2+}RT were seen when the hippocampal cells were Abeta(42)-treated. These effects were antagonized by BB treatment. Abeta(40) produced fewer deficits on Ca;{2+}RT than those seen when the HNC were pre-treated with either A;{2+}(42) or A;{2+}(25-35), but BB was relatively ineffective in antagonizing the deficits in Ca;{2+}RT produced by A;{2+}(40) or A;{2+}(25-35). Additional analyses indicated that BBs may be exerting their protective effects in the hippocampal cells by altering levels of phosphorylated MAPK, PKCgamma, and phosphorylated CREB. Therefore it appears that at least part of the protective effect of BBs may involve alterations in stress signaling.

Independence of synaptic specificity from neuritic guidance.

Neuronal circuits are interconnected with a high degree of specificity. While axonal guidance has been demonstrated to be crucial for the choice of the correct target region, its role in specificity at the level of individual cells remains unclear. Specificity of synapse formation may either result from precise guidance of axonal outgrowth onto the target or depend on a molecular "match" between pre- and postsynapse. To distinguish between these possibilities, an in vitro system was used in which neuritic outgrowth of rat cortical neurons is accurately guided along the narrow pathways of a surface micropattern. The micropattern consisted of a blend of extracellular matrix molecules applied to a cell repellent background of polystyrene by microcontact printing. The system reproduces guidance by attractant and repellent surface cues while no other signals that may influence synapse formation, like gradients of trophic factors or accumulations of signaling molecules, are provided. While the number of contact points between neighboring cells was strongly reduced on patterned substrates due to the geometrical restrictions, frequency of synapse formation was not different from homogeneous cultures. Thus it was unaffected by stringent guidance onto the target cell or by the number of cell-cell contacts. Moreover, a statistically significant enrichment of reciprocal contacts between mixed pairs of excitatory and inhibitory neurons over probabilistic predictions was found, which has similarly been shown by others in dissociated neuronal cultures. Our results indicate that precise axonal guidance is insufficient for target-specific synapse formation and suggest that instead recognition between individual cells is required.

Connectivity patterns in neuronal networks of experimentally defined geometry.

Experimental control over the position and connectivity pattern of neurons on a surface is of central interest for applications in biotechnology, such as cell-based biosensors and tissue engineering. By restricting neuronal networks to a simple grid pattern, a drastic reduction of network complexity can be achieved relative to networks on homogeneous substrates. Therefore, patterned neuronal networks are also a valuable tool in research on neuronal signal transduction. Microcontact printing has emerged as a simple and efficient method for surface patterning to direct cellular attachment. Although the formation of synaptic contacts in networks of rat cortical cells on such surfaces has been demonstrated, evidence of more complex circuits has been lacking. Triple patch-clamp measurements were performed to analyze connectivity in neuronal networks complying with a grid-shaped micropattern. Cells adhered stringently to the pattern and interconnected to a range of different types of circuits: linear connections, feedback loops, as well as branching and converging pathways. We conclude that in spite of the severe geometric restrictions, a complex repertoire of different connectivity patterns can form along the provided pathways. At the same time, network complexity is kept low enough to allow the study of these patterns at the resolution of single cell-cell contacts.

Copper deficiency as an anti-cancer strategy.

Copper is a tightly regulated trace element. Disruptions of copper homeostasis are rare and they cause serious disorders such as Wilson's disease and Menkes disease. Copper also plays an important role in promoting physiological and malignant angiogenesis. Formation of new blood vessels by a tumor enables tumor growth, invasion and metastasis. The copper chelator tetrathiomolybdate (TM), which quickly and effectively depletes copper stores, is under investigation as an anti-angiogenic agent. Promising results in vitro, in pre-clinical animal models and in an early (phase I) clinical trial have led to ongoing phase II evaluation of TM in patients with advanced cancers.

Patterning to enhance activity of cultured neuronal networks.

Embryonic rat hippocampal neurons were cultured in order to gain insights into how small networks of neurons interact. The principal observations are the electrical activities recorded with the electrode arrays, primarily action potentials both spontaneous and evoked. Several lithographic techniques were developed for controlling with micrometer precision the patterns of surface molecules in order to control neuronal attachment and growth. Cytophilic polylysine against protein repellent and hence cytophobic polyethylene glycol were used. By combining the cellular lithography with the microelectrode arrays it was possible to guide neurons preferentially to electrodes and to begin to investigate the question as to whether the geometric pattern of a neuronal network influences the patterns of its neuroelectric activity. It is clear that the techniques are adequate to ensure contact of neurons to electrodes but not to ensure the recording of signals, even when neurons lie directly on top of electrodes. The maturation of neuroelectric activity depends on the growth of glia within the culture, such that spontaneous activity appears to become robust when the number of glia is roughly the same as the number of neurons.

Higher respiratory rates and improved creatine stimulation in brain mitochondria isolated with anti-oxidants.

We tested the effect of an anti-oxidant mixture on respiration in isolated rat brain mitochondria. Mitochondria were isolated in mannitol/sucrose/EGTA/BSA +/- SCAVEGR anti-oxidants (SOD, catalase, vitamin E, vitamin E acetate, and glutathione reduced). TBARS were reduced by greater than 40% with SCAVEGR. Respiration driven by ADP showed a two-fold higher V(max) and a 15% higher respiratory control ratio when mitochondria were prepared with SCAVEGR. SCAVEGR also stabilized the octameric state of mitochondrial creatine kinase and thus improved creatine-stimulated respiration. These results suggest that significant improvements in brain mitochondrial function are obtained by isolation in the presence of an anti-oxidants mixture.

Tetrathiomolybdate anticopper therapy for Wilson's disease inhibits angiogenesis, fibrosis and inflammation.

The need for agents to lower body copper in Wilson's disease, a disease which results from copper toxicity has been the driving force for the development of the effective anticopper drugs penicillamine, trientine, zinc, and now tetrathiomolybdate (TM). Because of its rapid action, potency, and safety, TM is proving to be a very effective drug for initial treatment of acutely ill Wilson's disease patients. Beyond this, TM has antiangiogenic effects, because many proangiogenic cytokines require normal levels of copper. This has led to use of TM in cancer, where it is generally effective in animal tumor models, and has shown efficacy in preliminary clinical studies. Most recently, it has been found that TM has antifibrotic and antiinflammatory effects through inhibition of profibrotic and proinflammatory cytokines.

Age-related differences in NMDA responses in cultured rat hippocampal neurons.

The N-methyl-D-aspartate receptor (NMDAR) is expressed in the cerebral cortex and hippocampus and is important in learning and memory. NMDARs are influenced by aging and implicated in neurodegenerative disorders. We investigated age-related differences in NMDAR ionic currents and intracellular calcium in embryonic (E18), middle-age (9-10 month) and old (26 month) rat hippocampal neurons cultured in serum-free medium for 7-12 days. Responses to 200 microM NMDA with 50 microM glycine were measured using whole cell voltage clamp and fura-2 fluorescence. Embryonic neurons exhibited significantly larger and faster NMDA responses than adults. Old rats had 1.5 fold greater normalized NMDA peak current compared to middle-age rats, while intracellular calcium rose 1.3 fold higher. Differences in regression slopes generated from the integral of NMDA current versus normalized NMDA current indicate age-related differences are not exclusively due to changes in receptor density but likely influenced by changes in receptor function. Corresponding age-related measures of intracellular calcium by fura-2 fluorescence in response to NMDA showed a strong correlation with peak current (r(2)=0.996). Our data support the hypothesis that NMDAR responsiveness is altered during aging with an enhanced NMDA peak current in both old and embryonic neurons compared to middle-age neurons.

Zinc acetate for the treatment of Wilson's disease.

Zinc acetate (Galzin, Gate Pharmaceutical Co.) has been developed for the treatment of Wilson's disease, an inherited disease of copper accumulation and copper toxicity in brain and liver. Zinc acetate has been approved by the US FDA for maintenance therapy of adult and paediatric Wilson's disease patients but also has efficacy in the treatment of pregnant patients and presymptomatic patients from the beginning. It also has value as adjunctive therapy for the initial treatment of symptomatic patients. Zinc's mechanism of action involves induction of intestinal cell metallothionein (Mt), which blocks copper absorption from the intestinal track. A negative copper balance is caused by blockade not only of absorption of food copper but the blockade of reabsorption of the considerable amount of endogenously secreted copper in saliva, gastric juice and intestinal secretions. Zinc is completely effective in controlling copper levels and toxicity in Wilson's disease, as are other anticopper agents. Zinc's major advantage over other anticopper agents is its extremely low level of toxicity. The only side effect is some degree of initial gastric irritation in approximately10% of patients, which usually decreases and becomes insignificant over time. As with all long-term therapies, compliance is a problem in some patients and dictates regular monitoring with 24 h urine copper and zinc measurements. As with all anticopper therapies, over a long period of time, overtreatment and induction of copper deficiency can occur. This is to be avoided particularly in children because copper is required for growth.

Modulation of neural network activity by patterning.

Using neuronal cultures on microelectrode arrays, researchers have shown that recordable electrical activity can be influenced by chemicals in the culture environment, thus demonstrating potential applicability to biosensors or drug screening. Since practical success requires the design of robust networks with repeatable, reliable responses understanding the sources of variation is important. In this report, we used lithographic technologies to confine neurons to highly defined patterns (40 microm wide stripes); in turn these patterns gave us a measure of control over the local density of neurons (100-500 cells/mm(2)). We found that the apparent electrical activity of the network, as measured by the fraction of electrodes from which signals were recordable, increases 8-10-fold with greater local density. Also, average-firing rates of the active neurons increased 3-5-fold. We conclude that patterned networks offer one means of controlling and enhancing the responsiveness of cultured neural networks.

Copper control as an antiangiogenic anticancer therapy: lessons from treating Wilson's disease.

The search for new anticopper drugs for Wilson's disease is culminating in two excellent new drugs: zinc for maintenance therapy and tetrathiomolybdate (TM) for initial therapy. Both are effective and nontoxic. TM is a very potent, fast-acting new anticopper drug and its properties may be useful well beyond Wilson's disease. Angiogenesis (new blood vessel growth) is required for tumor growth, and a sufficient level of copper appears to be required for angiogenesis. We hypothesize that there is a "window" to which the copper level can be reduced that inhibits angiogenesis in tumors, but does not interfere with vital cellular functions of copper. Using TM therapy, this approach has worked to slow or stabilize tumor growth in several animal tumor models, and preliminary results are also very encouraging in human patients with a variety of advanced and metastatic malignancies. A hypothesis is advanced that copper availability has played a fundamental role in growth regulation throughout evolution and that is the reason that so many angiogenic promoters appear to be dependent upon copper levels.

Age- and concentration-dependent neuroprotection and toxicity by TNF in cortical neurons from beta-amyloid.

The induction of an inflammatory response and release of cytokines such as TNF may be involved in the age-related etiology of Alzheimer disease (AD). In the brain, microglia have been shown to produce a wide variety of immune mediators, including the pro-inflammatory cytokine tumor necrosis factor (TNF). We hypothesize that with age there is increased ability of microglia to produce TNF or that age decreases the neuroprotective effect of TNF against beta-amyloid (Abeta) toxicity in neurons. We investigated the effects of Abeta(1-40) on TNF secretion from forebrain cultures of microglia from embryonic, middle-age (9-month) and old (36-month) rats. Over the first 12 hr of exposure to 10 microM Abeta (1-40), microglia from embryonic and old rats increase TNF secretion, although microglia from middle-age rats did not produce detectable levels of TNF. When low concentrations of TNF are added to neurons together with Abeta (1-40) in the absence of exogenous antioxidants, neuroprotection for old neurons is significantly less than neuroprotection for middle-age neurons. In neurons from old rats, high levels of TNF together with Abeta are more toxic than in neurons from middle-age or embryonic rats. These results are discussed in relation to neuroprotection and toxicity of the age-related pathology of AD.

Culture and regeneration of human neurons after brain surgery.

Cortical human brain tissue was obtained from 11 craniotomies for intractable epilepsy or tumor resection. Neuregen transport medium preserved viability at 4 degrees C during transfer to the culture laboratory. Cells were isolated and cultured by methods previously developed for adult rat neurons (Brewer GJ. Isolation and culture of adult rat hippocampal neurons. J. Neurosci. Meth. 1997:71:143-55). In about 40% of the cases, cultures regenerated with a majority of neuron-like cells that stained for neurofilament and not GFAP. After 3 weeks of culture from a 70 year old meningioma case, synapse-like structures were revealed by electron microscopy. Trophic support from basic human recombinant fibroblast growth factor was synergistically improved with the steroid hormone dehydroepiandrosterone 3-sulfate. Another 40% of the cases resulted in cultures that were predominantly GFAP positive astroglia. The remaining 20% of the cases did not regenerate cells with neuron-like or glial processes. Three postmortem cases did not regenerate neurites. These methods may aid development of human culture models of epilepsy as well as human pharmacology, toxicology and development of improved methods for brain grafts.

Temperature and time interval for culture of postmortem neurons from adult rat cortex.

For a model of neurological disease and ischemia, we extended recent work to culture adult postmortem rat brain neurons. Frontal cortex sections were removed from adult rats immediately following sacrifice and at different postmortem intervals and with the brain at either 22 degrees C or 4 degrees C. Brain could be stored four times longer at 4 degrees C between sacrifice and neuronal disaggregation to achieve the same 20% recovery of live cells from those plated compared to 22 degrees C. Each milligram of rat frontal cortex was estimated by the optical disector method to contain 160,000 neurons. When cells were isolated as rapidly as possible, 9% of the neurons originally present in the brain were viable. Various postmortem intervals from 2 to 24 hr resulted in a reduction from 6% to 3% of the cells originally present. After 5 days in culture, viable neurons were 23-42% of those isolated. Neuron-like cells that survived represented 40-75% of the viable cells, or 0.5-2.75% of those originally estimated to be present in the brain. Electrophysiology experiments show that cells isolated 0 and 24 hr postmortem had neuronal electrical properties, including an average resting membrane potential of -48 mV, voltage-sensitive currents, and action potentials. Neuron-like cells were immunoreactive for neuron-specific enolase, neurofilament 200, glutamate, MAP2, and tau after 2 weeks in culture. These experiments show that neuron-like cells can be reliably cultured from adult rat cortex up to 6 hr postmortem when stored at 22 degrees C and up to 24 hr postmortem when stored at 4 degrees C. These findings should encourage donation of human postmortem brain neurons for studies on ischemia, adult pharmacology, and neurological disease.