John D. C. Lambert

Epileptiform activity in combined slices of the hippocampus, subiculum and entorhinal cortex during perfusion with low magnesium medium ☆

Reduction of [Mg2+]o induced spontaneous epileptiform activity consisting of 40-100-ms bursts of population spikes in hippocampal slices. This activity disappeared from area CA1 when the connections to area CA3 were cut, but persisted in isolated minislices of area CA3. Spontaneous activity was also observed in the dentate gyrus, provided that the connections to the subiculum and entorhinal cortex (EC) were intact. In the parasubiculum and EC longer lasting epileptiform events were observed which resembled seizure-like behaviour. The epileptiform activity was completely suppressed by 2-aminophosphonovalerate (30 microM) suggesting that N-methyl-D-aspartate receptors for excitatory amino acid transmitters participate in the generation of this activity. These findings show that the EC possesses properties which permit the generation of seizure-like activity in contrast to the hippocampus where the activity resembled recurrent interictal events.

The slow inhibitory postsynaptic potential in rat hippocampal CA1 neurones is blocked by intracellular injection of QX-314.

Intracellular recordings were made from CA1 pyramidal neurones in the rat hippocampus slice preparation. The recording electrodes contained potassium acetate (4 M) with or without the quaternary lidocaine derivative, QX-314 (50 mM). Both fast (f) and slow (s) inhibitory postsynaptic potentials (IPSP) were evoked by low-frequency orthodromic stimulation. The s-IPSP was rapidly reduced by QX-314 injection. It decreased along a similar time course to the dV/dt of the action potential (AP). The f-IPSP and excitatory postsynaptic potential were not significantly reduced in size at a time when the s-IPSP was virtually abolished by QX-314. It is concluded that conductance through the K+ channels which are coupled to GABAB receptors is readily blocked by QX-314, while the Cl- channels which are coupled to GABAA receptors and the cation channels coupled to the glutamate receptors are relatively resistant to the local anaesthetic.

Effects of new non‐N‐methyl‐D‐aspartate antagonists on synaptic transmission in the in vitro rat hippocampus.

1. 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 1.2 and 4.8 microM, respectively. In Mg2+‐free solutions responses to NMDA were generally not affected by concentrations of CNQX up to 25 microM. 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 CA1 and CA3 neurones with an IC50 of around 2 microM, 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 (greater than 5 microM) a small EPSP remained which was largest in CA1 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 CA1 was the same as the CNQX‐sensitive EPSP, indicating that NMDA receptors participate in monosynaptic excitation. 7. Feedback and feed‐forward inhibition in both area CA1 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 CNQX‐resistant IPSP was not affected by APV. 8. In conclusion, quinoxalinediones have been used to demonstrate that non‐NMDA receptors mediate the majority of the EPSP. Additionally, a component of the EPSP in CA1 is mediated by NMDA receptors and is manifested at resting membrane potentials and in the presence of Mg2+.

Regenerative properties of pyramidal cell dendrites in area CA1 of the rat hippocampus.

1. Intracellular recordings were obtained from 184 distal apical dendrites and twenty‐six somata of CA1 pyramidal neurones in the rat hippocampal slice preparation. In the presence of 3.25 mM K+ 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 38.8% 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 one‐third 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 (82.2 +/‐ 1.9 mV) and their rate of rise (81.3 +/‐ 4.3 V s‐1) was markedly slower than that of somatic spikes (291.5 +/‐ 17.8 V s‐1). 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 Ca(2+)‐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 microM). The secondary slow spikes within the complex were blocked by Co2+ (2 mM), nifedipine (10 microM) and high concentrations (> 50 microM) of verapamil, while Ni2+ (100‐300 microM) had no effect. Thus, compound spiking consists of an initial Na(+)‐dependent spike followed by one or more slow Ca(2+)‐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.(ABSTRACT TRUNCATED AT 400 WORDS)

GABAB receptors play a major role in paired-pulse facilitation in area CA1 of the rat hippocampus.

Extracellular recordings of field potentials in area CA1 of the rat hippocampal slice have been used to investigate paired-pulse facilitation. Field potentials were evoked by maximal stimulation of the Schaffer collateral/commissural fibres. The height of the population spike (PS) in stratum pyramidale (str. pyr.) and the area under the field excitatory postsynaptic potential (EPSP) following the PS in the stratum radiatum (str. rad.) were quantified. These values were used to describe the time course of paired-pulse facilitation. Facilitation of the PS was maximal 50 ms after the conditioning pulse and was present over a period of about 500 ms. However, facilitation of the late area (LA) of the field EPSP was maximal afer 125 ms and had an overall duration of 1-2 s. The N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonovaleric acid (APV), had no effect on paired-pulse facilitation of either the LA or the PS. The gamma-aminobutyric acid-B (GABAB) agonist baclofen increased facilitation of the PS. This was mainly due to a reduction of the unconditioned response. Facilitation of the LA was reduced by both baclofen and the GABAB antagonist, 2-OH-saclofen. Baclofen increased the LA of the unconditioned response, while this was unaffected by 2-OH-saclofen. The LA of facilitated responses was decreased by 2-OH-saclofen while the effect of baclofen on these responses was more complex. Baclofen reduced the LA of maximally facilitated responses, while the LA of slightly facilitated responses was increased. The results show that different mechanisms are involved in the facilitation of the LA and the PS. Furthermore, activation of GABAB receptors makes a large contribution to paired-pulse facilitation of the field EPSP. It is also suggested that recording of extracellular fields in str. rad. in response to paired-pulse stimulation provides a simple electrophysiological model for testing the effect of agents which act at the GABAB receptor.

Somatic amplification of distally generated subthreshold EPSPs in rat hippocampal pyramidal neurones

1 Intracellular recordings from hippocampal CA1 pyramidal neurones revealed that EPSPs evoked by selective stimulation of the isolated afferent input to the distal third of the apical dendrites were relatively insensitive to changes in dendritic membrane potential (Vm) but amplified by depolarizations of the somatic Vm. The amplification was present at potentials depolarized from resting membrane potential (RMP) but was most marked when the EPSPs were close to threshold for action potential generation. The amplification consisted of a uniform component and a variable component which was only present when the EPSPs were threshold straddling. 2 The somatic amplification was caused by an intrinsic membrane current which was blocked by somatic application of tetrodotoxin (TTX, 10 μm), but was insensitive to bath application of NiCl2 (100–200 μm). We therefore suggest that the amplification of the subthreshold EPSP is due primarily to the activation of a non‐inactivating Na+ current (INaP). 3 Injection of 4‐aminopyridine (4‐AP, 25–50 mM) during intradendritic recordings resulted in amplification of the EPSPs in 37 % of the dendrites, which was similar to that observed in somatic recordings. However, in the one case in which somatic application of TTX was tested, dendritic amplification was blocked, suggesting that it is a reflection of the somatic amplification. 4 Because the shift to variable amplification was very abrupt and it is present in only a very narrow voltage range close to threshold, we suggest that the variable component is caused by the regenerative activation of INaP. The variability itself is probably due to the simultaneous activation of different outward K+ currents. 5 The present results indicate that the somatic region of CA1 pyramidal neurones can function as a voltage‐dependent amplifier of distally evoked EPSPs and that this is due to the activation of a somatic INaP. The presence of this amplifying mechanism will have important functional consequences for the way in which distally generated EPSPs are integrated.

Comparison of the effect of the GABA uptake blockers, tiagabine and nipecotic acid, on inhibitory synaptic efficacy in hippocampal CA1 neurones

The action of the novel gamma-aminobutyric acid (GABA) uptake blocker, tiagabine, has been studied on isolated GABAergic fast inhibitory postsynaptic potentials (IPSP) and currents (IPSC) in rat hippocampal CA1 pyramidal cells in the slice preparation. Tiagabine (20-50 microM) had little effect on the peak amplitude of the IPSC, but caused a robust increase in the half-width (by 109 +/- 15%). These results contrasted with those obtained using the established uptake blocker, nipecotic acid (100 microM to 1 mM), which reduced the amplitude of the IPSC by 35 +/- 6% and caused only a modest prolongation of the recovery phase. These effects, which were poorly reversible, are probably explained by the fact that nipecotic acid is a substrate for the GABA-uptake carrier and can act as a false transmitter. Tiagabine is not transported by the GABA carrier and results with this substance demonstrate the role of uptake in determining the kinetics of activation of GABAA receptors. Tiagabine is proposed as the blocker of choice for the GABA uptake system.

5,10,15,20-Tetrakis(N-Methyl-4-Pyridyl)-21H,23H-Porphine (TMPyP) as a Sensitizer for Singlet Oxygen Imaging in Cells: Characterizing the Irradiation-dependent Behavior of TMPyP in a Single Cell†

Abstract Singlet molecular oxygen, a1Δg, can be detected from a single cell by its weak 1270 nm phosphorescence (a1Δg→X3Σg−) upon irradiation of the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) incorporated into the cell. The behavior of this sensitizer in a cell, and hence the behavior of the associated singlet oxygen phosphorescence signal, depends on the conditions under which the sample is exposed to light. Upon irradiation of a neuron freshly incubated with TMPyP, the intensity of TMPyP fluorescence initially increases and there is a concomitant increase in the singlet oxygen phosphorescence intensity from the cell. These results appear to reflect a photoinduced release of TMPyP bound to DNA in the nucleus of the cell, where TMPyP tends to localize, and the subsequent relocalization of TMPyP to a different microenvironment in the cell. Upon prolonged irradiation of the cell, TMPyP photobleaches and there is a corresponding decrease in the singlet oxygen phosphorescence intensity from the cell. The data reported herein provide insight into key factors that can influence photosensitized singlet oxygen experiments performed on biological samples.

Factors determining the efficacy of distal excitatory synapses in rat hippocampal CA1 pyramidal neurones

1 A new preparation of the in vitro rat hippocampal slice has been developed in which the synaptic input to the distal apical dendrites of CA1 pyramidal neurones is isolated. This has been used to investigate the properties of distally evoked synaptic potentials. 2 Distal paired‐pulse stimulation (0.1 Hz) evoked a dendritic response consisting of a pair of EPSPs, which showed facilitation. The first EPSP had a rise time (10‐90 %) of 2.2 ± 0.05 ms and a half‐width of 9.1 ± 0.13 ms. The EPSPs were greatly reduced by CNQX (10 μm) and the remaining component could be enhanced in Mg2+‐free Ringer solution and blocked by AP5 (50 μm). In 70 % of the dendrites, the EPSPs were followed by a prolonged after‐hyperpolarizarion (AHP) which could be blocked by a selective and potent GABAB antagonist, CGP 55845A (2 μm). These results indicate that the EPSPs are primarily mediated by non‐NMDA receptors with a small contribution from NMDA receptors, whereas the AHP is a GABAB receptor‐mediated slow IPSP. 3 With intrasomatic recordings, the rise time of proximally generated EPSPs (3.4 ± 0.1 ms) was half that of distally generated EPSPs (6.7 ± 0.5 ms), whereas the half‐widths were similar (19.6 ± 0.8 ms and 23.8 ± 1 ms, respectively). These results indicate that propagation through the proximal apical dendrites slows the time‐to‐peak of distally generated EPSPs. 4 Distal stimulation evoked spikes in 60 % of pyramidal neurones. At threshold, the distally evoked spike always appeared on the decaying phase of the dendritic EPSP, indicating that the spike is initiated at some distance proximal to the dendritic recording site. Furthermore, distally and proximally generated threshold spikes had a similar voltage dependency. These results therefore suggest that distally generated threshold spikes are primarily initiated at the initial segment. 5 At threshold, spikes generated by stimulation of distal synapses arose from the decaying phase of the dendritic EPSPs with a latency determined by the time course of the EPSP at the spike initiation zone. With maximal stimulation, however, the spikes arose directly from the peak of the EPSPs with a time‐to‐spike similar to the time‐to‐peak of subthreshold dendritic EPSPs. Functionally, this means that the effect of dendritic propagation can be overcome by recruiting more synapses, thereby ensuring a faster response time to distal synaptic inputs. 6 In 42 % of the neurones in which distal EPSPs evoked spikes, the relationship between EPSP amplitude and spike latency could be accounted for by a constant dendritic modulation of the EPSP. In the remaining 58 %, the change in latency was greater than can be accounted for by a constant dendritic influence. This additional change in latency is best explained by a sudden shift in the spike initiation zone to the proximal dendrites. This would explain the delay observed between the action of somatic application of TTX (10 μm) on antidromically evoked spikes and distally evoked suprathreshold spikes. 7 The present results indicate that full compensation for the electrotonic properties of the main proximal dendrites is not achieved despite the presence of Na+ and Ca2+ currents. Nevertheless, distal excitatory synapses are capable of initiating spiking in most pyramidal neurones, and changes in EPSP amplitude can modulate the spike latency. Furthermore, even though the primary spike initiation zone is in the initial segment, the results suggest that it can move into the proximal apical dendrites under physiological conditions, which has the effect of further shortening the response time to distal excitatory synaptic inputs.

Direct Optical Detection of Singlet Oxygen from a Single Cell

Abstract Singlet oxygen has been detected in single nerve cells by its weak 1270 nm phosphorescence (a1Δg→X3Σg−) upon irradiation of a photosensitizer incorporated in the cell. Thus, one can now consider the application of direct optical imaging techniques to mechanistic studies of singlet oxygen at the single-cell level.