RESUMEN
Neurons and dynamical models of spike generation display two different types of threshold behavior, with steady current stimulation: type 1 [the firing frequency vs. current (f-I) relationship is continuous at threshold) and type 2 (discontinuous f-I)]. The dynamics at threshold can have profound effects on the encoding of input as spikes, the sensitivity of spike generation to input noise, and the coherence of population firing. We have examined the f-I and frequency-conductance (f-g) relationships of cells in layer 2/3 of slices of young (15-21 DIV) rat somatosensory cortex, focusing in detail on the nature of the threshold. Using white-noise stimulation, we also measured firing frequency and interspike interval variability as a function of noise amplitude. Regular-spiking (RS) pyramidal neurons show a type 1 threshold, consistent with their well-known ability to fire regularly at very low frequencies. In fast-spiking (FS) inhibitory interneurons, although regular firing is supported over a wide range of frequencies, there is a clear discontinuity in their f-I relationship at threshold (type 2), which has not previously been highlighted. FS neurons are unable to support maintained periodic firing below a critical frequency fc, in the range of 10 to 30 Hz. Very close to threshold, FS cells switch irregularly between bursts of periodic firing and subthreshold oscillations. These characteristics mean that the dynamics of RS neurons are well suited to encoding inputs into low-frequency firing rates, whereas the dynamics of FS neurons are suited to maintaining and quickly synchronizing to gamma and higher-frequency input.
Asunto(s)
Neuronas/fisiología , Umbral Sensorial/fisiología , Corteza Somatosensorial/fisiología , Potenciales de Acción/fisiología , Algoritmos , Animales , Electrofisiología , Técnicas In Vitro , Modelos Neurológicos , Red Nerviosa/fisiología , Conducción Nerviosa/fisiología , Neuronas/clasificación , Ratas , Ratas Wistar , Receptores AMPA/fisiología , Corteza Somatosensorial/citología , Membranas Sinápticas/fisiologíaRESUMEN
LC-MS and LC-MS/MS analyses were used to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, (-)-(1R,2S,3S,4R)-1,2-epoxy-3,4-dihydroxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene [(-)-(R,S,S,R)-BcPh DE-2], on reaction in vitro with an oligonucleotide dodecamer derived from the HPRT gene. The sequence of this dodecamer, 5'-T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12)-3', contains a base (corresponding to A(7)) which is a hot spot for mutagenesis in the hprt gene induced by the carcinogenic (R,S,S,R)-enantiomer of benzo[a]pyrene 7,8-diol 9,10-epoxide, and an adjacent base (corresponding to A(6)) which gave no mutations with this diol epoxide. Modified oligonucleotides were generated by reaction of (-)-BcPh DE-2 with both the single-stranded and duplex forms of the dodecamer. Multiple purine targets in both strands led to the formation of complex reaction mixtures of regioisomeric BcPh DE-modified oligonucleotides, which were partially separated by reverse phase HPLC on a polystyrene-divinylbenzene column. On-line LC-MS data allowed facile distinction between adducts on the two strands of the duplex, and MS/MS analysis permitted unambiguous assignment of the major sites of modification in the regioisomeric, adducted strands. In the duplex, these sites were at A(6), A(7), and G(8). Interestingly, the "hot spot" A(7)w as about 3 times more reactive with the BcPh DE than the "cold spot" A(6). Adduct formation from the single-stranded dodecamer was less selective, and resulted in more extensive alkylation of G residues.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Aductos de ADN/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , Fenantrenos/análisis , EstereoisomerismoRESUMEN
Neurons in the functioning cortex fire erratically, with highly variable intervals between spikes. How much irregularity comes from the process of postsynaptic integration and how much from fluctuations in synaptic input? We have addressed these questions by recording the firing of neurons in slices of rat visual cortex in which synaptic receptors are blocked pharmacologically, while injecting controlled trains of unitary conductance transients, to electrically mimic natural synaptic input. Stimulation with a Poisson train of fast excitatory (AMPA-type) conductance transients, to simulate independent inputs, produced much less variability than encountered in vivo. Addition of NMDA-type conductance to each unitary event regularized the firing but lowered the precision and reliability of spikes in repeated responses. Independent Poisson trains of GABA-type conductance transients (reversing at the resting potential), which simulated independent activity in a population of presynaptic inhibitory neurons, failed to increase timing variability substantially but increased the precision of responses. However, introduction of synchrony, or correlations, in the excitatory input, according to a nonstationary Poisson model, dramatically raised timing variability to in vivo levels. The NMDA phase of compound AMPA-NMDA events conferred a time-dependent postsynaptic variability, whereby the reliability and precision of spikes degraded rapidly over the 100 msec after the start of a synchronous input burst. We conclude that postsynaptic mechanisms add significant variability to cortical responses but that substantial synchrony of inputs is necessary to explain in vivo variability. We suggest that NMDA receptors help to implement a switch from precise firing to random firing during responses to concerted inputs.
Asunto(s)
Potenciales de Acción/fisiología , Vías Aferentes/metabolismo , Corteza Cerebral/metabolismo , Sincronización Cortical , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Vías Aferentes/citología , Animales , Corteza Cerebral/citología , Estimulación Eléctrica , Técnicas In Vitro , Inhibición Neural/fisiología , Neuronas/citología , Ratas , Ratas Wistar , Receptores AMPA/metabolismo , Sinapsis/ultraestructura , Factores de TiempoRESUMEN
Allylamine was pulse-plasma polymerized onto a hydrophobic polysiloxane substrate to create cell adhesion surfaces for cell culture that would not require pretreatment with polylysine, could be sterilized via autoclaving, and could be re-used for several culture cycles. We investigated two different plasma deposition protocols at 200 W RF power: (1) a duty cycle of 3 ms on and 5 ms off; and (2) a cycle of 3 ms on and 45 ms off. Control surfaces were unmodified polysiloxane, activated polysiloxane via flaming, and flamed polysiloxane further modified with poly-D-lysine (PDL). The different surfaces were characterized with XPS analysis, water contact angle, and cell adhesion and growth using dissociated murine embryonic spinal tissue. We found that both the amine content of the 3/45 duty cycle surface and the wettability was higher than that of the 3/5 surface. Also, spinal cord cells were better dispersed 24 h after seeding on the 3/45 surface, suggesting a difference in early adhesion dynamics. However, the networks on the two types of modified surfaces revealed no obvious morphological differences after 2 weeks in vitro. The stability of allylamine-decorated surfaces after autoclaving was high with only minor changes in wettability and nitrogen content. Cell growth on such surfaces after autoclaving was comparable to that found on flamed polysiloxane, freshly modified with PDL. Allylamine surfaces were still usable as cell growth substrates after three autoclaving cycles, 4 weeks under warm culture medium, and simple cleaning procedures, indicating the achievement of a long-lasting modification that did not require the repeated use of PDL before each seeding.
Asunto(s)
Alilamina/química , Técnicas de Cultivo de Célula/métodos , Neuronas/citología , Siloxanos/química , Alilamina/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Estabilidad de Medicamentos , Feto/citología , Calor , Ratones , Ratones Endogámicos BALB C , Polímeros , Siloxanos/farmacología , Médula Espinal/citología , Esterilización/métodos , Propiedades de SuperficieRESUMEN
Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.
Asunto(s)
Bleomicina/análogos & derivados , Daño del ADN , Espectrometría de Masas/métodos , Oligonucleótidos/química , Secuencia de Bases , Bleomicina/química , Bleomicina/farmacología , ADN/química , ADN/efectos de los fármacos , ADN/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico/efectos de los fármacosRESUMEN
Identification and quantitation of covalent carcinogen-DNA adducts, an important class of biomarkers, is an integral goal in toxicological research. Since these adducts are commonly present at very low levels in in vivo samples, sensitive and specific analytical methodologies are imperative for accurate detection, characterization and quantitation. High-performance separations coupled to electrospray mass spectrometry (ESI-MS) provide the sensitivity and specificity required for the analysis of DNA adducts. This review provides an overview over the research conducted in this area, focusing on the application of HPLC-ESI-MS and CE-ESI-MS techniques for structural analysis and quantitation of modified nucleosides, nucleotides and oligonucleotides.
Asunto(s)
Aductos de ADN/análisis , Espectrometría de Masas/métodos , Cromatografía Líquida de Alta Presión , Electroforesis CapilarRESUMEN
This paper describes a novel method to map guanine bases in short oligonucleotides using a simple chemical modification reaction and subsequent analysis by electrospray ionization ion trap mass spectrometry (ITMS). In situ guanine-specific methylation followed by gas-phase fragmentation permits the determination of the positions of all guanine residues. Collision-induced dissociation (CID) of the monomethylated oligonucleotide strand promotes rapid depurination and further collision (MS3) of the apurinic oligonucleotide leads to preferential cleavage of the backbone at the site of depurination. The mass of the resulting complementary product ions verifies the position of each guanine base in the sequence. The utility of this methodology is demonstrated for oligonucleotide sequences up to 10 bases in length. In addition, this technique successfully illustrates the use of selective fragmentation for sequencing oligonucleotides by ITMS.
Asunto(s)
Espectrometría de Masas , Oligonucleótidos/química , Guanina/química , Espectrometría de Masas/métodos , MetilaciónRESUMEN
The stability of plasma-polymerized allylamine films with autoclaving sterilization cycles was investigated. Polymerized films were deposited under pulsed plasma conditions using two different duty cycles to provide surfaces having different initial amino group concentrations. The film properties were analyzed by XPS and water contact angle measurements before and after autoclaving. The reactions of these surfaces with trifluoroacetic anhydride provided quantitation of the amino surface concentrations before and after autoclaving. In general, the plasma-polymerized films exhibit good stability vis à vis the autoclaving process, with relatively high retention of the surface amino groups. The results of this work are of specific value with respect to tissue culture studies in which surface modifications involving the introduction of amino groups have been shown to have high efficacy in promoting cell growth. The results obtained suggest that the simple one-step plasma treatment process is a viable alternative to the more cumbersome surface modification procedures currently employed to introduce amino groups in these tissue culture studies.
Asunto(s)
Alilamina/análogos & derivados , Alilamina/química , Materiales Biocompatibles/química , Anhídridos Acéticos , Estabilidad de Medicamentos , Fluoroacetatos , Calor , Esterilización/métodos , Propiedades de Superficie , TermodinámicaRESUMEN
We studied the effect of a novel neurotoxin, alpha-pompilidotoxin (alpha-PMTX) on the spontaneously synchronized network firing of cultured rat cortical neurons. Alpha-PMTX acted immediately and irreversibly to disrupt synchronous activity, leaving only residual sparse, uncorrelated firing and was effective at concentrations of 10 nM. In the presence of bicuculline to block inhibitory synaptic transmission, the shutdown in synchronized activity occurred with a significant delay, required a higher concentration of alpha-PMTX (> 100 nM), and was preceded by a transiently increased level of firing. It appears that both inhibitory and excitatory neuronal activity or synaptic transmission are amplified by alpha-PMTX, but that intense activity eventually leads to inactivation or transmitter depletion.
Asunto(s)
Corteza Cerebral/citología , Neuronas/efectos de los fármacos , Neurotoxinas/farmacología , Transmisión Sináptica/efectos de los fármacos , Venenos de Avispas/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Células Cultivadas , Proteínas de Insectos , Vías Nerviosas/efectos de los fármacos , Neuronas/citología , Ratas , Transmisión Sináptica/fisiologíaRESUMEN
We introduce a new member of a family of photochemically active oligonucleotide conjugates. A Phenanthrodihydrodioxin (PDHD)-based agent was synthesized and covalently linked to a 5'-end of the 9-mer oligonucleotide via a hexamethylene linker. The conjugate hybridized to a complementary 30-nucleotide-long target and efficiently cleaved it in a sequence specific manner. Up to 67% of target was specifically damaged (51% cross-links and 16% direct cleavage). While the photosensitizer alone nonspecifically damaged only Gs in a single-stranded target, its conjugate cross-linked to and damaged also A, T, and C sites in a target in agreement with duplex and triplex formation.
Asunto(s)
ADN Viral/metabolismo , Dioxinas/metabolismo , Fármacos Fotosensibilizantes/química , Bacteriófago M13/química , Reactivos de Enlaces Cruzados/metabolismo , Luz , Estructura Molecular , Hibridación de Ácido Nucleico , Oligonucleótidos/química , Fenantrenos/química , Quinonas/química , Análisis de Secuencia de ADN , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
This work demonstrates the interfacing of capillary electrophoresis in a poly(N-vinylpyrrolidone) (PVP) solution to electrospray ionization ion trap mass spectrometry (ESI-ITMS). This methodology was used for on-line analysis of modified and unmodified oligonucleotides. Oligonucleotides were covalently modified using the model carcinogen 2-(N-acetoxy-N-acetylamino)fluorene. In the presence of PVP, separation was achieved for a set of isomeric (N-acetylamino)fluorene (AAF)-modified oligonucleotides differing only in their base sequences, while open-tube control experiments showed no separation between these compounds. The resolved analytes were identified by ESI-ITMS with negative ion detection. Online acquisition of MS and MS/MS data allowed unambiguous identification of all structural isomers. Baseline separation was also accomplished for a 10-compound mixture containing a series of five nonisomeric AAF-modified oligonucleotides and their unmodified parent oligonucleotides.
Asunto(s)
Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Oligonucleótidos/química , 2-Acetilaminofluoreno , CarcinógenosAsunto(s)
Anemia de Células Falciformes/complicaciones , Bronquitis/etiología , Dolor en el Pecho/complicaciones , Disnea/complicaciones , Enfermedad Aguda , Bronquitis/diagnóstico , Bronquitis/patología , Broncoscopía , Niño , Preescolar , Tos/complicaciones , Femenino , Fiebre/complicaciones , Humanos , Lactante , Masculino , SíndromeRESUMEN
Neurons, by virtue of intrinsic electrophysiological mechanisms, represent transducers that report the dynamics of cell death, receptor-ligand interactions, alterations in metabolism, and generic membrane perforation processes. In cell culture, mammalian neurons form fault-tolerant, spontaneously active systems with great sensitivity to their chemical environment and generate response profiles that are often concentration- and substance-specific. Changes in action potential patterns are usually detected before morphological changes and cell damage occur, which provides sensitivity and reversibility. Such biological systems can be used to screen rapidly for novel pharmacological substances, toxic agents, and for the detection of certain odorants. Existing simple culture preparations can already be employed effectively for the detection of chemical compounds. So far, three strategies have been investigated in pilot experiments: (1) Substance-dependent major changes in spontaneous native activity patterns. All synaptically active agents (e.g. glutamate, strychnine, N-methyl D-aspartic acid) as well as metabolic poisons generate such changes. (2) Substance-dependent changes in network oscillations via disinhibition. The regularized, oscillatory activity is altered by synaptically and metabolically active substances, ion channel blockers, and toxins. (3) Detection of paroxysmal responses indicating major, pathological membrane currents in large subpopulation of cells. We have explored these three strategies via 64 channel array recordings using spontaneously active murine spinal cord cultures. The glycine receptor blocker strychnine reliably generated increased multichannel bursting at 5-20 nM and regular, coordinated bursting above 5 microM. During biculline-induced network oscillations many compounds alter oscillation frequencies or terminate activity in a substance-specific manner. Finally, the gp120 protein of the AIDS virus (at 1 microgram/ml) produces massive, unique paroxysmal discharges that may last as long as 2 min. These results indicate that cultured neuronal networks are practical systems that can be used for the detection and identification of a great variety of chemical substances. The concept of dynamic fingerprinting to identify specific compounds is discussed.
Asunto(s)
Técnicas Biosensibles , Red Nerviosa/efectos de los fármacos , Animales , Células Cultivadas , Ratones , Red Nerviosa/fisiología , Reproducibilidad de los Resultados , Estricnina/farmacología , TemperaturaRESUMEN
Neurons, by virtue of intrinsic electrophysiological mechanisms, represent transducers that report the dynamics of cell death, receptor-ligand interactions, alterations in metabolism and generic membrane perforation processes. In cell culture, mammalian neurons form fault-tolerant, spontaneously active systems with great sensitivity to their chemical environment and generate response profiles that are often concentration- and substance-specific. Changes in action potential patterns are usually detected before morphological changes and cell damage occur. This provides sensitivity and reversibility. Such biological systems may be used to screen rapidly for novel pharmacological substances, toxic agents and certain odorants. This paper reports on substance-dependent major changes in spontaneous native activity patterns by the synaptically active (glycine receptor blocker) strychnine. Via 64-channel array recordings of spontaneously active murine spinal cord cell cultures, increased multichannel bursting at 5-20 nM strychnine and regular, coordinated bursting above 5 microM could be reliably generated. By artificial neural network analysis a quantitative correlation of network signals and strychnine concentration could be evaluated for small concentrations of strychnine. The results indicate that cultured neuronal networks already represent reliable and practical systems which can be used for the detection of chemical substances and the characterization of their biological influences.
Asunto(s)
Técnicas Biosensibles , Convulsivantes/farmacología , Red Nerviosa/fisiología , Estricnina/farmacología , Animales , Células Cultivadas , Ratones , Red Nerviosa/efectos de los fármacos , Médula Espinal/citología , Médula Espinal/fisiologíaRESUMEN
The protein product of the cystic fibrosis (CF) gene, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is known to function as an apical chloride channel at the surface of airway epithelial cells. It has been proposed that CFTR has additional intracellular functions and that there is altered processing of mutant forms. In examining these functions we found a stable form of CFTR with slow turnover in surface membrane preparations from CF and non-CF immortalized airway epithelial cell lines. The methods used to study the turnover of CFTR were pulse/chase experiments utilizing saturation labeling of [35S] Met with chase periods of 5-24 h in the presence of 8 mM Met and cell fractionation techniques. Preparations of morphologically identifiable surface membranes were compared to total cell membrane preparations containing intracellular membranes. Surface membrane CFTR had lower turnover defined by pulse/chase ratios than that of the total cell membrane preparations. Moreover, mutant CFTR was stable in the surface membrane fraction with little degradation even after a 24 h chase, whereas wild-type CFTR had a higher pulse/chase ratio at 24 h. In the presence of 50 microM castanospermine, which is an inhibitor of processing alpha-glucosidases, a more rapid turnover of mutant CFTR was found in the total cell membrane preparation, whereas wild-type CFTR had a lower response. The results are compatible with a pool of CFTR in or near the surface membranes which has an altered turnover in CF and a glycosylation-dependent alteration in the processing of mutant CFTR.