Steen Nedergaard

Nifedipine‐ and omega‐conotoxin‐sensitive Ca2+ conductances in guinea‐pig substantia nigra pars compacta neurones.

1. The membrane properties of substantia nigra pars compacta (SNc) neurones were recorded in guinea‐pig in vitro brain slices. 2. In the presence of tetrodotoxin (TTX) a Ca(2+)‐dependent slow oscillatory potential (SOP) was generated. Application of 0.5‐20 microM nifedipine abolished both spontaneous and evoked SOPs. A tetraethylammonium chloride (TEA)‐promoted high‐threshold Ca2+ spike (HTS) was little affected by nifedipine. On the other hand, omega‐conotoxin applied either locally or via the perfusion medium (1‐10 microM) blocked a part of the HTS, but it did not alter the SOP. 3. In normal medium nifedipine blocked the spontaneous discharge, decreased the interspike interval (ISI) recorded during depolarizing current injections and selectively reduced the slow component of the spike after‐hyperpolarization (AHP). omega‐Conotoxin decreased both the rising and falling slopes of the normal action potential, reduced the peak amplitude of the spike AHP, and, in some of the neurones, reduced the ISI during depolarization. The Na+ spikes recorded in Ca(2+)‐free medium were not altered by omega‐conotoxin. 4. The SOP was not blocked by octanol (100‐200 microM), amiloride (100‐250 microM), or Ni2+ (100‐300 microM). However, at 500 microM Ni2+ attenuated the SOP. 5. Application of apamin (0.5‐2.0 microM) induced irregular firing or bursting, abolished the slow component of the spike AHP and reduced its peak amplitude. In the presence of TTX and apamin long‐duration plateau potentials occurred, which were subsequently blocked by nifedipine. 6. In Ca(2+)‐free, Co(2+)‐containing medium TTX‐sensitive spikes and voltage plateaux were generated by depolarizing current pulses. It is suggested that a persistent Na+ conductance underlies the plateaux, which may be co‐activated during the SOP. 7. The results suggest that the Ca2+ currents underlying the SOP and the HTS are different and that they activate at least two Ca(2+)‐dependent K+ conductances. These conductances play major roles in the maintenance of spontaneous discharge and in control of membrane excitability.

The modulation of excitatory amino acid responses by serotonin in the cat neocortex in vitro

Summary1.The electrophysiological actions of excitatory amino acids and serotonin were investigated in slices from cat neocortexin vitro. Intracellular recordings were obtained from neurons (mainly in layer V) and the drugs applied extracellularly to the same neurons by microiontophoresis.2.Serotonin, and to some extent noradrenaline, facilitated the excitatory actions ofN-methyl-d-aspartate (NMDA), glutamate, and quisqualate but caused no changes in the passive neuronal membrane properties when presented alone. Serotonin had no effect on evoked excitatory postsynaptic potentials (EPSPs) or spike afterhyperpolarizations.3.The facilitatory effect of serotonin on the responses to NMDA was observed with both somatic and dendritic applications. It persisted during Mg2+ depletion and in the presence of tetrodotoxin and tetraethylammonium. The effect was attenuated by the serotonin antagonist cinanserin but not by methysergide. A possible underlying receptor modulation is discussed.

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.

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.

Differential influence of non-synaptic mechanisms in two in vitro models of epileptic field bursts

Non-synaptic interactions are known to promote epileptiform activity through mechanisms that have primarily been studied in one particular in vitro model (low Ca(2+) model). Here we characterize another non-synaptic model, where ictal-like field bursts are induced in the CA1 area of rat hippocampal slices by exposure to Cs(+) (4-5mM) together with blockers of fast chemical synaptic transmission, and compare it with the low Ca(2+) model. The Cs-induced field bursts were blocked by 1 microM tetrodotoxin, but persisted in the presence of 200 microM Cd(2+) or 300 microM Ni(2+). Hyperosmotic condition (addition of 30 mM sucrose), reduced burst amplitude, but, unlike field bursts induced by 0mM Ca(2+)/5.25 mM K(+), sucrose had no effect on frequency or duration. Intracellular alkalinization-acidification sequence induced by NH(4)Cl potentiated and blocked, respectively, the field bursts. Octanol (100-250 microM) blocked all activity in most experiments. A quantitative comparison of three gap junction antagonists (carbenoxolone (100 microM), quinidine (100-250 microM), and endothelin-3 (1-2 microM)) indicated that gap junction communication is implicated in both models. However, endothelin-3 had selective effect on the low Ca(2+)-induced field burst. The data suggest that extracellular space-dependent processes, including field effects, significantly contribute to ongoing field burst activity, whereas initiation of a field burst can occur with or without the aid of such interactions, depending on the level of neuronal excitability. Gap junctions seem to have a general role in initiating field bursts. However, the contribution to this effect from neuronal versus glial connexin types differs in the two epileptic models studied.

Cognitive deficits caused by a disease-mutation in the α3 Na+/K+-ATPase isoform

The Na+/K+-ATPases maintain Na+ and K+ electrochemical gradients across the plasma membrane, a prerequisite for electrical excitability and secondary transport in neurons. Autosomal dominant mutations in the human ATP1A3 gene encoding the neuron-specific Na+/K+-ATPase α3 isoform cause different neurological diseases, including rapid-onset dystonia-parkinsonism (RDP) and alternating hemiplegia of childhood (AHC) with overlapping symptoms, including hemiplegia, dystonia, ataxia, hyperactivity, epileptic seizures, and cognitive deficits. Position D801 in the α3 isoform is a mutational hotspot, with the D801N, D801E and D801V mutations causing AHC and the D801Y mutation causing RDP or mild AHC. Despite intensive research, mechanisms underlying these disorders remain largely unknown. To study the genotype-to-phenotype relationship, a heterozygous knock-in mouse harboring the D801Y mutation (α3+/D801Y) was generated. The α3+/D801Y mice displayed hyperactivity, increased sensitivity to chemically induced epileptic seizures and cognitive deficits. Interestingly, no change in the excitability of CA1 pyramidal neurons in the α3+/D801Y mice was observed. The cognitive deficits were rescued by administration of the benzodiazepine, clonazepam, a GABA positive allosteric modulator. Our findings reveal the functional significance of the Na+/K+-ATPase α3 isoform in the control of spatial learning and memory and suggest a link to GABA transmission.

A novel mechanism for the anticonvulsant effect of furosemide in rat hippocampus in vitro

Though both in vivo and in vitro studies have demonstrated an anticonvulsant effect of the loop diuretic furosemide, the precise mechanism behind this effect is still debated. The current study investigates the effect of furosemide on Cs-induced epileptiform activity (Cs-FP) evoked in area CA1 of rat hippocampal slices in the presence of Cs(+) (5mM) and ionotropic glutamatergic and GABAergic receptor antagonists. As this model diverges in several respects from other epilepsy models it can offer new insight into the mechanism behind the anticonvulsive effect of furosemide. The present study shows that furosemide suppresses the Cs-FP in a dose-dependent manner with a near complete block at concentrations ≥ 1.25 mM. Because furosemide targets several types of ion transporters we examined the effect of more selective antagonists. Bumetanide (20 μM), which selectively inhibits the Na-K-2Cl co-transporter (NKCC1), had no significant effect on the Cs-FP. VU0240551 (10 μM), a selective antagonist of the K-Cl co-transporter (KCC2), reduced the ictal-like phase by 51.73 ± 8.5% without affecting the interictal-like phase of the Cs-FP. DIDS (50 μM), a nonselective antagonist of Cl(-)/HCO3(-)-exchangers, Na(+)-HCO3(-)-cotransporters, chloride channels and KCC2, suppressed the ictal-like phase by 60.8 ± 8.1% without affecting the interictal-like phase. At 500 μM, DIDS completely suppressed the Cs-FP. Based on these results we propose that the anticonvulsant action of furosemide in the Cs(+)-model is exerted through blockade of the neuronal KCC2 and Na(+)-independent Cl(-)/HCO3(-)-exchanger (AE3) leading to stabilization of the activity-induced intracellular acidification in CA1 pyramidal neurons.

Suppression of epileptiform activity by a single short-duration electric field in rat hippocampus in vitro

The mechanisms behind the therapeutic effects of electrical stimulation of the brain in epilepsy and other disorders are poorly understood. Previous studies in vitro have shown that uniform electric fields can suppress epileptiform activity through a direct polarizing effect on neuronal membranes. Such an effect depends on continuous DC stimulation with unbalanced charge. Here we describe a suppressive effect of a brief (10 ms) DC field on stimulus-evoked epileptiform activity in rat hippocampal brain slices exposed to Cs(+) (3.5 mM). This effect was independent of field polarity, was uncorrelated to changes in synchronized population activity, and persisted during blockade of synaptic transmission with Cd(2+) (500 μM). Antagonists of A(1), P(2X), or P(2Y) receptors were without effect. The suppressive effect depended on the alignment of the external field with the somato-dendritic axis of CA1 pyramidal cells; however, temporal coincidence with the epileptiform activity was not essential, as suppression was detectable for up to 1 s after the field. Pyramidal cells, recorded during epileptiform activity, showed decreased discharge duration and truncation of depolarizing plateau potentials in response to field application. In the absence of hyperactivity, the applied field was followed by slow membrane potential changes, accompanied by decreased input resistance and attenuation of the depolarizing afterpotential following action potentials. These effects recovered over a 1-s period. The study suggests that a brief electric field induces a prolonged suppression of epileptiform activity, which can be related to changes in neuronal membrane properties, including attenuation of signals depending on the persisting Na(+) current.

The slow Ca2+ -dependent K+ -current facilitates synchronization of hyperexcitable pyramidal neurons.

Studies on in vivo and in vitro epilepsy models have shown that progression and maintenance of epileptiform activity can be affected by the slow Ca(2+)-dependent K(+) current (I(sAHP)). This study aimed to investigate the influence of the I(sAHP) on population activity and single cell activity during the transition from the interictal- to the ictal-like phase of an epileptiform field potential induced by Cs(+). Extracellular and intracellular recordings were performed in area CA1 on 400 microm thick hippocampal slices from adult male Wistar rats. During maintained exposure to Cs(+), the transition between the two phases underwent a slow, time-dependent change, where synchronized population activity gradually disappeared and a plateau-like prolongation of the interictal-like phase emerged. In parallel, the size of the ictal-like phase increased. These effects could be ascribed to a gradual block of the I(sAHP) and were mimicked by the I(sAHP) antagonists carbacholine (2 microM), isoproterenol (4 microM) and Ba(2+) (0.2 mM). Cessation of population activity generally occurred without a concomitant decrease in firing rate of single CA1 pyramidal neurons, but was accompanied by the disappearance of hyperpolarizing prepotentials, indicating a shift in the mechanism of action potential initiation. These findings suggest that the presence of the I(sAHP) increases the tendency of hyperexcitable neurons to fire in synchrony, but at the same time serves to dampen the ictal-like activity that follows the hyperexcitable state. Our data indicate that both effects can be attributed to the influence of this current on the steady-state membrane potential in the period of the transition from interictal- to ictal-like activity.

Opposing effects of 2-deoxy-D-glucose on interictal- and ictal-like activity when K+ currents and GABAA receptors are blocked in rat hippocampus in vitro.

The ketogenic diet (KD), a high-fat, carbohydrate-restricted diet, is used as an alternative treatment for drug-resistant epileptic patients. Evidence suggests that compromised glucose metabolism has a significant role in the anticonvulsant action of the KD; however, it is unclear what part of the glucose metabolism that is important. The present study investigates how selective alterations in glycolysis and oxidative phosphorylation influence epileptiform activity induced by blocking K+ currents and GABAA and NMDA receptors in the hippocampal slice preparation. Blocking glycolysis with the glucose derivative 2-deoxy-d-glucose (2-DG; 10 mM) gave a fast reduction of the frequency of interictal discharge (IED) consistent with findings in other in vitro models. However, this was followed by the induction of seizure-like discharges in area CA1 and CA3. Substituting glucose with sucrose (glucopenia) had effects similar to those of 2-DG, whereas substitution with l-lactate or pyruvate reduced the IED but had a less proconvulsant effect. Blockade of ATP-sensitive K+ channels, glycine or adenosine 1 receptors, or depletion of the endogenous anticonvulsant compound glutathione did not prevent the actions of 2-DG. Baclofen (2 μM) reproduced the effect of 2-DG on IED activity. The proconvulsant effect of 2-DG could be reproduced by blocking the oxidative phosphorylation with the complex I toxin rotenone (4 μM). The data suggest that inhibition of IED, induced by 2-DG and glucopenia, is a direct consequence of impairment of glycolysis, likely exerted via a decreased recurrent excitatory synaptic transmission in area CA3. The accompanying proconvulsant effect is caused by an excitatory mechanism, depending on impairment of oxidative phosphorylation. NEW & NOTEWORTHY This study reveals two opposing effects of 2-deoxy-d-glucose (2-DG) and glucopenia on in vitro epileptiform discharge observed during combined blockade of K+ currents and GABAA receptors. Interictal-like activity is inhibited by a mechanism that selectively depends on impairment of glycolysis and that results from a decrease in the strength of excitatory recurrent synaptic transmission in area CA3. In contrast, 2-DG and glucopenia facilitate ictal-like activity by an excitatory mechanism, depending on impairment of mitochondrial oxidative phosphorylation.