In time- and spatially resolved experiments, singlet molecular oxygen, O(2)(a(1)Delta(g)), was created in a single nerve cell upon irradiation of a sensitizer incorporated in the cell nucleus using a focused laser beam. The singlet oxygen thus produced was detected by its infrared phosphorescence. Data obtained indicate that, contrary to common perception, this reactive species can be quite long-lived in a cell and, as such, can diffuse over appreciable distances including across the cell membrane into the extra-cellular environment. These results provide a new perspective for mechanistic studies of photoinduced cell death and intracellular signaling.

The effect of low extracellular Mg2+ concentration ([Mg2+]o) on neuronal activity was studied in rat hippocampal slices. After 20-40 min of perfusion with Mg2+-free medium, when [Mg2+]o declined to approximately 0.1-0.4 mM, spontaneous field potentials developed in the CA1 and CA3 regions, but not in the dentate gyrus. In the CA3 pyramidal cell layer, these potentials consisted of repetitive (0.3-0.5 Hz), 40- to 120-ms-long positive deflections (2-5 mV) with superimposed population spikes. In the stratum (str.) pyramidale of the CA1 region, positive-negative deflections (less than 3 mV) lasting for 30-80 ms were observed, which occurred with a frequency of 0.3-0.5 Hz. In some cases, longer lasting and rapidly recurring events were also observed. In CA3 pyramidal cells, the intracellular correlates of the field potential transients were 20- to 30-mV paroxysmal depolarization shifts (PDS) with superimposed bursts of action potentials, followed by large (greater than 10 mV), 500- to 1,200-ms-long afterhyperpolarizations (AHP). In contrast, pyramidal neurons of the CA1 area did not show PDSs; instead, sequences of excitatory postsynaptic potentials (EPSPs)/inhibitory postsynaptic potentials (IPSPs) accompanied the transient field potential changes. Occasionally, spontaneous EPSPs/IPSPs, occurring with high frequencies, could also be observed in CA1 without any field potential transients. In both hippocampal regions, the epileptiform activity evolved without significant alterations in the resting membrane potential (RMP) and input resistance (RN) of the neurons, although a 2- to 5-mV reduction in action potential threshold was noted. The spontaneous activity in Mg2+-free medium was readily suppressed by raising the extracellular Ca2+ concentration ([Ca2+]o) from 1.6 to 3.6 mM. The perfusion of 10-30 microns DL-2-amino-5-phosphonovaleric acid (2-APV), an antagonist for the glutamate receptors of the N-methyl-D-aspartate (NMDA) type, also attenuated or reversibly blocked the spontaneous activity. Surgical isolation of area CA1 from CA3 ceased the occurrence of the transients in CA1 but not in CA3. The synaptic input/output curves were shifted to the left in the absence of [Mg2+]o. Threshold intensity for eliciting population spikes was 50-75% of that in normal medium. Paired-pulse facilitation was still present near threshold, but was reduced at higher stimulus intensities. Decreases in [Ca2+]o, produced by repetitive stimulation (20-Hz/5-10 s) of the Schaffer collateral/commissural pathway and monitored with ion-selective microelectrodes in the CA1 region, were enhanced in Mg2+-free medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Singlet molecular oxygen, O(2)(a(1)Delta(g)), has been detected from single neurons and HeLa cells in time-resolved optical experiments by its 1270 nm phosphorescence (a(1)Delta(g)--> X(3)Sigma(-)(g)) upon irradiation of a photosensitizer incorporated into the cell. The cells were maintained in a buffered medium and their viability was assessed by live/dead assays. To facilitate the detection of singlet oxygen, intracellular H(2)O was replaced with D(2)O by an osmotic de- and rehydration process. The effect of this insult on the cells was likewise assessed. The data indicate that, in the complicated transition from a "live" to "dead" cell, the majority of our cells have the metabolic activity and morphology characteristic of a live cell. Quenching experiments demonstrate that the singlet oxygen lifetime in our cells is principally determined by interactions with intracellular water and not by interactions with other cell constituents. The data indicate that in a viable, metabolically-functioning, and H(2)O-containing cell, the lifetime of singlet oxygen is approximately 3 micros. This is consistent with our previous reports, and confirms that the singlet oxygen lifetime in a cell is much longer than hitherto believed. This implies that, in a cell, singlet oxygen is best characterized as a selective rather than reactive intermediate. This is important when considering roles played by singlet oxygen as a signaling agent and as a component in events that result in cell death. The data reported herein also demonstrate that spatially-resolved optical probes can be used to monitor selected events in the light-induced, singlet-oxygen-mediated death of a single cell.

The lowest excited electronic state of molecular oxygen, singlet molecular oxygen, O(2)(a (1)Delta(g)), is a reactive species involved in many chemical and biological processes. To better understand the roles played by singlet oxygen in biological systems, particularly at the sub-cellular level, optical tools have been developed to create and directly detect this transient state in time- and spatially-resolved experiments from single cells. Data obtained indicate that, contrary to common perception, this reactive species can be quite long-lived in a cell and, as such, can diffuse over appreciable distances including across the cell membrane into the extracellular environment. On one hand, these results demonstrate that the behavior of singlet oxygen in an intact cell can be significantly different from that inferred from model bulk studies. More generally, these results provide a new perspective for mechanistic studies of intra- and inter-cellular signaling and events that ultimately lead to photo-induced cell death.

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In time-resolved and spatially resolved experiments, singlet molecular oxygen, O2(a1Deltag), was created in a single nerve cell upon irradiation of a sensitizer incorporated in the cell using a focused laser beam. The singlet oxygen thus produced was detected by its infrared phosphorescence. Data obtained indicate that in both the cytoplasm and the nucleus of the cell, this reactive species is approximately 1-2 orders of magnitude longer-lived than previously believed. The data demonstrate that deactivation of singlet oxygen in the cell is dominated by interactions with the solvent not cellular constituents such as proteins. These results provide a new perspective for mechanistic studies of the role of O2(a1Deltag) in photoinduced cell death and intracellular signaling.

1. Intracellular recordings were obtained from 184 distal apical dendrites and twenty-six somata of CAI pyramidal neurones in the rat hippocampal slice preparation. In the presence of 3-25 mm KF 200 ms suprathreshold current pulses evoked three different types of firing patterns in the apical dendrites, all of which were distinct from regular somatic firing. Fast tetrodotoxin (TTX)-sensitive spiking was evoked in 38f8% of the dendrites. Compound spiking, consisting of an initial fast spike followed by one or more secondary slow spikes of variable amplitude and duration, was seen in 44-1 % of dendrites. 'Classical' burst firing, resembling intrinsic somatic bursts, was evoked in 17-1 % of the dendrites. 2. In fast spiking dendrites, the spikes evoked by long depolarizing pulses were rarely overshooting, showed prominent accommodation and declined progressively to about onethird of the initial amplitude. The amplitude of single dendritic fast spikes (50-6 + 1-5 mV; mean + S.E.M.) was smaller than that of somatic spikes (82X2 ± 1X9 mV) and their rate of rise (81X3 + 4-3 V s-') was markedly slower than that of somatic spikes (291-5 + 17-8 V s-').However, the thresholds were not significantly different (dendrites, -49-8 + 0-8 mV; somata, -50-8 + 1-3 mV). These results indicate that fast spikes in the distal parts of apical dendrites are generated by a local regenerative Na+ current. 3. 4-Aminopyridine (4-AP, 0-1-0'5 mM) caused a dose-dependent slowing of the repolarization of the fast spikes, while tetraethylammonium (TEA, 2 mM) and Co2+ (2 mM) induced a slowing of the late phase of the repolarization. These results indicate that the transient outward K+ current, IA' and the Ca2+-activated K+ current, Ic, are involved in the repolarization of dendritic Na+-dependent spikes.4. Compound spiking was completely blocked by TTX (0 5-1 juM). The secondary slow spikes within the complex were blocked by Co2+ (2 mM), nifedipine (10 /SM) and high concentrations (>50 juM) of verapamil, while Ni2+ (100-300 ,uM) had no effect. Thus, compound spiking consists of an initial Na+-dependent spike followed by one or more slow Ca2+-dependent spikes mediated by L-type Ca2+ channels located in the apical dendrites. 5. In fast spiking dendrites, 4-AP (0-5-2-5 mM) changed the firing pattern from regular fast spiking to compound spiking. In the presence of 4-AP (0 1-0 5 mM), the single fast spike evoked by a short (20 ms), threshold current pulse, was followed by secondary slow spikes of variable amplitude and duration. This suggests that activation of a dendritic IA has a significant influence on the spatial activation ofdendritic L-type Ca2+ channels.6. Increasing the extracellular K+ concentration to 5-25 mm had no effect on the passive membrane properties of the dendrites. However, the incidence of bursting dendrites increased to 83%, while the remaining 17% showed compound spiking. The burst responses were blocked by TTX (0 5-1 SM), while Ni2+ (100-300 UM), nifedipine (10 fuM) and verapamil (50-100 uM) had no effect, indicating that neit...

SUMMARY1. The effects of new, potent non-N-methyl-D-aspartate (NMDA) receptor antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), have been examined using intra-and extracellular recordings in the hippocampal slice preparation. In terms of potency and selectivity, the action of the two blockers was similar and CNQX was used in most experiments.2. CNQX reduced the responses to ionophoretic applications of the non-NMDA agonists kainate (KAI) and quisqualate (QUIS) with IC50 values of 1P2 and 4-8 am, respectively. In Mg2+-free solutions responses to NMDA were generally not affected by concentrations of CNQX up to 25 /tM.3. The action of CNQX was only slowly and poorly reversible on washing. Responses to QUIS and KAI were also reversibly reduced by ionophoretic application of CNQX.4. CNQX blocked the evoked EPSP in CAI and CA3 neurones with an IC5 of around 2 /tM, which is similar to the IC50 for responses to KAI. CNQX was without effect on the passive membrane properties, the afferent volley and paired pulse potentiation. 5. In the presence of CNQX (> 5 /tM) a small EPSP remained which was largest in CAI neurones. It was blocked by low concentrations of the NMDA receptor antagonist (± )-2-amino-5-phosphonovaleric acid (APV), was markedly enhanced on removing Mg2`ions from the bathing medium and, in voltage-clamp experiments, showed a potential dependence which is characteristic of the NMDA ionophore.6. The latency of the APV-sensitive EPSP in CAI was the same as the CNQXsensitive EPSP, indicating that NMDA receptors participate in monosynaptic excitation.7. Feedback and feed-forward inhibition in both area CAl and CA3 were sensitive to CNQX. There seemed to be two components of the inhibition, both of which appear to be GABAergic since they could be blocked by picrotoxin (PTX), but only one of which was blocked by CNQX. The