Mogens Andreasen

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)

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.

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 demonstration of an receptor mediated component of excitatory synaptic transmission in area CA1 of the rat hippocampus

The action of a new non-N-methyl-D-aspartate (NMDA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), on synaptic transmission in area CA1 of the rat hippocampus has been examined. Intracellular and extracellular recordings showed CNQX to be a potent antagonist of synaptic potentials evoked by stimulation of the Schaffer collateral-commissural fibre system. One to 2 microM CNQX was sufficient to reduce the excitatory postsynaptic potential (EPSP) by 50%. CNQX is therefore about 100 times more potent than previously available non-NMDA receptor antagonists. In the presence of CNQX, a small depolarizing potential could still be evoked. This potential was sensitive to the NMDA-receptor blocker, 2-amino-5-phosphonovaleric acid (APV), increased in size on depolarizing the neurone and also increased in size on removing Mg2+ from the perfusing medium. This residual EPSP therefore has characteristics which are consistent with its mediation via the NMDA receptor-coupled ionophore. These results indicate a dual composition of the monosynaptic excitatory potential in area CA1.

Dendritic electrogenesis in rat hippocampal CA1 pyramidal neurons: functional aspects of Na+ and Ca2+ currents in apical dendrites.

The regenerative properties of CA1 pyramidal neurons were studied through differential polarization with external electrical fields. Recordings were obtained from somata and apical dendrites in the presence of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), DL‐2‐amino‐5‐phosphonovaleric acid (APV), and bicuculline. S+ fields hyperpolarized the distal apical dendrites and depolarized the rest of the cell, whereas S÷ fields reversed the polarization. During intradendritic recordings, S+ fields evoked either fast spikes or compound spiking. The threshold response consisted of a low‐amplitude fast spike and a slow depolarizing potential. At higher field intensities the slow depolarizing potential increased in amplitude, and additional spikes of high amplitude appeared. During intrasomatic recordings, S+ field evoked repetitive firing of fast spikes, whereas S÷ fields evoked a slow depolarizing, potential on top of which high‐ and low‐amplitude spikes were evoked. Tetrodotoxin (TTX) blocked all types of responses in both dendrites and somata. Perfusion with Ca2+‐free, Co2+‐containing medium increased the frequency and amplitude of fast spikes evoked by S+ field and substantially reduced the slow depolarizing potential evoked by S÷ fields. Antidromic stimulation revealed that an all‐or‐none dendritic component was activated in the distal apical dendrites by back‐propagating somatic spikes. The dendritic component had an absolute refractory period of about 4 ms and a relative refractory period of 10–12 ms. Ca2+‐dependent spikes in the dendrites were followed by a long‐lasting afterhyperpolarization (AHP) and a decrease in membrane input resistance, during which dendritic excitability was selectively reduced. The data suggest that generation of fast Na+ currents and slow Ca2+ currents in the distal part of apical dendrites is highly sensitive to the dynamic state of the dendritic membrane. Depending on the mode and frequency of activation these currents can exert a substantial influence on the input‐output behavior of the pyramidal neurons.

Noradrenaline receptors participate in the regulation of GABAergic inhibition in area Ca1 of the rat hippocampus

1. Standard intracellular recordings from CA1 pyramidal neurones in in vitro hippocampal slices have been used to investigate the effects of excitatory amino acid antagonists and adrenergic agents on evoked synaptic potentials. 2. Ortho‐ and antidromic stimulation were conducted with remotely placed electrodes in order to minimize the possibility of stimulating the interneurones directly. In addition to the excitatory postsynaptic potential (EPSP), orthodromic stimulation evoked an inhibitory sequence consisting of a fast and slow inhibitory postsynaptic potential (IPSP). The slow‐IPSP was blocked by intracellular injection of QX 314. Antidromic stimulation evoked a relatively pure fast‐IPSP. 3. In seven neurones the differential effects of glutamatergic receptor blockers on the fast‐IPSP were investigated. The N‐methyl‐D‐aspartate (NMDA) receptor blocker, DL‐2‐amino‐5‐phosphonovaleric acid (APV) was added after the full effect of the non‐NMDA receptor blocker, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) had been achieved. In three neurones, APV had no additional blocking effect, while in the remaining four neurones, both the ortho‐ and antidromically evoked IPSPs were reduced by 20‐50%. This suggests that NMDA receptors participate in the activation of some GABAergic interneurones, which was further confirmed by showing that the IPSP was enhanced by Mg(2+)‐free medium. 4. In the presence of CNQX (10 microM) and APV (50 microM) together, the ortho‐ and antidromically evoked fast‐IPSPs were greatly reduced. A small ‘residual’ IPSP remained which was best studied by depolarizing the neurone to around ‐50 mV. With maximum stimulation, this amounted to 26.3 +/‐ 15.4% (mean +/‐ S.E.M., n = 15) of the control IPSP evoked by orthodromic stimulation and 41 +/‐ 14.6% of the control IPSP evoked by antidromic stimulation. The following statements apply equally to the ortho‐ and antidromically activated residual IPSPs. 5. The residual IPSP was completely blocked by low concentrations of bicuculline, indicating that it is mediated by GABAA receptors. When compared with a control IPSP of similar amplitude, the residual IPSP was found to have a faster rise time and time‐to‐peak, but a similar decay time. 6. Neither the muscarinic cholinergic antagonist, atropine nor the presynaptic glutamate agonist, L‐2‐amino‐4‐phosphonobutyric acid (L‐APB) had any effect on the residual IPSP. 7. The residual IPSP was completely blocked by the adrenergic beta‐receptor antagonist, L‐propranolol (50‐100 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of the kinetics of synaptic inhibition points to a reduction in GABA release in area CA1 of the genetically epileptic mouse, El

In order to determine whether changes in synaptic inhibition are involved in chronic models of epilepsy, it is necessary to understand the factors which determine the kinetics of fast gamma-aminobutyric acid (GABA)ergic inhibition. For this purpose, we analyzed the decaying phase of isolated inhibitory postsynaptic currents (IPSC) in rats CA1 pyramidal cells. Reduction of GABA release (by reducing [Ca2+]o or paired-pulse stimulation) or blockade of GABA uptake (with tiagabine) led to the conclusion that small changes in the amount of GABA available for postsynaptic binding have little effect on the peak amplitude, but have marked effect on the duration of the IPSC. We then studied isolated GABAA receptor-mediated inhibition in area CA1 of the El mouse strain, which is genetically predisposed to epilepsy. Results were compared with the non-epileptogenic mother strain, ddY. Inhibitory postsynaptic potentials (IPSPs) in El mice (IPSPEl) were not significantly different in amplitude of those from ddY mice (IPSPddY). However, the rise-time and duration of IPSPEl were respectively about 25% and 50% shorter than those of IPSPddY. With appropriate pharmacological manipulation of GABA release or uptake, IPSPEl could be made to resemble the IPSPddY and vice versa. It is concluded that the synaptic release of GABA in area CA1 of the El mouse is decreased compared to that of the ddY mouse.

The role of excitatory amino acids in synaptic transmission in the hippocampus

We have highlighted some aspects of the action of excitatory amino acid transmission in the hippocampus. Fast epsps can be blocked by CNQX to reveal a component of synaptic transmission which is mediated by NMDA receptors. Extracellular recordings of ionic activities show that NMDA and non-NMDA ionophores are permeable to the major monovalent cations, while NMDA ionophores also appear to be permeable to Ca2+. Interactions of agonists applied by iontophoresis may be correlates of phenomena such as LTP, which can be evoked by appropriate synaptic stimulation.

Influence of the hyperpolarization-activated cation current, Ih, on the electrotonic properties of the distal apical dendrites of hippocampal CA1 pyramidal neurones

The electrical field application technique has revealed that the electrotonic length of the distal apical dendrites of hippocampal CA1 pyramidal neurones is long compared to the rest of the cell. This difference may be due to an asymmetrical distribution of channels responsible for the leak conductance in distal and proximal membrane segments. One such conductance, the hyperpolarization-activated cation current, I(h), is reported to display an increasing density with distance from the soma along the apical dendrite. Such asymmetry of I(h) could be a major cause of the increased electrotonic length of the distal apical dendrite. In the present study we found that blockade of I(h), by bath application of Cs(+) (3 mM) or ZD7288 (20 microM), reduced the electrical field-induced transmembrane polarization (TMP) in the distal apical dendrites. In some neurones the polarization reversed polarity, reflecting a movement of the indifference point (site of zero polarization) from the distal dendrites, across the recording site to a more proximal position. These effects were more pronounced when Cs(+) and ZD7288 were applied locally to the distal apical dendrites. Bath application of another antagonist of leak conductance, Ba(2+) (1 mM), also decreased the average field-induced polarization. This latter effect, however, did not reach statistical significance. These data suggest that I(h) is partly responsible for the distal location of the indifference point, and indicate that an elevated activity of I(h) contributes to the relatively increased electrotonic length of the most distal part of the apical dendrites.