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Arachidonic acid (AA) is a free fatty acid membrane-permeable second messenger that is liberated from cell membranes via receptor- and Ca2+-dependent events. We have shown previously that extremely low [AA]i (1 pm) inhibits the postsynaptic voltage-gated K+ current (IA) in hippocampal neurons. This inhibition is blocked by some antioxidants. The somatodendritic IA is mediated by Kv4.2 gene products, whereas presynaptic IA is mediated by Kv1.4 channel subunits. To address the interaction of AA with these α-subunits we studied the modulation of A-currents in human embryonic kidney 293 cells transfected with either Kv1.4 or Kv4.2 rat cDNA, using whole-cell voltage-clamp recording. For both currents 1 pm [AA]i inhibited the conductance by > 50%. In addition, AA shifted the voltage dependence of inactivation by −9 (Kv1.4) and +6 mV (Kv4.2), respectively. Intracellular co-application of Trolox C (10 μm), an antioxidant vitamin E derivative, only slowed the effects of AA on amplitude. Notably, Trolox C shifted the voltage dependence of activation of Kv1.4-mediated IA by −32 mV. Extracellular Trolox for > 15 min inhibited the AA effects on IA amplitudes as well as the effect of intracellular Trolox on the voltage dependence of activation of Kv1.4-mediated IA. Extracellular Trolox further shifted the voltage dependence of activation for Kv4.2 by +33 mV. In conclusion, the inhibition of maximal amplitude of Kv4.2 channels by AA can explain the inhibition of somatodendritic IA in hippocampal neurons, whereas the negative shift in the voltage dependence of inactivation apparently depends on other neuronal channel subunits. Both AA and Trolox potently modulate Kv1.4 and Kv4.2 channel α-subunits, thereby presumably tuning presynaptic transmitter release and postsynaptic somatodendritic excitability in synaptic transmission and plasticity. [ABSTRACT FROM AUTHOR]

Oxidative stress affects cellular membrane lipids and proteins. Using whole-cell patch-clamp recording we demonstrate differential oxidative inhibition of voltage-gated transient (IA) and delayed rectifier [IK(V)] K+ currents by arachidonic acid (AA) and H2O2 in CA1 neurons in hippocampal slice. We show that intracellular application of 1 pm AA or its non-metabolizable analog eicosatetraynoic acid (100 pm) reduced IA by ∼42% but did not affect IK(V). AA shifted the voltage dependence of steady-state inactivation of IA by 12 mV to more negative potentials whereas the rate of inactivation was unchanged. Surprisingly, intracellular glutathione (GSH, 20 mm) enhanced the effect of AA on maximal IA (−62%) and with AA slowed inactivation of IA. The combination of GSH and extracellular ascorbate (0.4 mm) prevented reduction of IA by AA. Intracellular Trolox (a vitamin E analog, 10 µm) reduced IA by 61%and IK(V) by 39%. Like AA, intracellular Trolox caused a 10-mV left shift of IA steady-state inactivation but Trolox and AA did not cause a shift when coapplied. Extracellular Trolox (100 µm) had no effects on IA. H2O2 (80 µm) reduced both IA and IK(V) in a GSH- and ascorbate-sensitive manner and slowed the rate of inactivation of IA by a factor of 2. Coapplication of H2O2 with GSH and extracellular ascorbate caused ∼22 mV negative shifts of both steady-state inactivation and activation. We conclude that AA is extremely potent in affecting IA by oxidative modifications. Antioxidants can augment these effects, probably by catalysis of the underlying reactions between oxidants and IA channel proteins. [ABSTRACT FROM AUTHOR]

Cholinergic activation of entorhinal cortex (EC) layer V neurons plays a crucial role in the medial temporal lobe memory system and in the pathophysiology of temporal lobe epilepsy. Here, we demonstrate that muscarinic activation by focal application of carbachol depolarizes EC layer V neurons and induces epileptiform activity in rat brain slices. These seizure-like bursts are associated with a somatic [Ca2+]i increase of 293 ± 82 n m and are blocked by the glutamate receptor antagonists CNQX and APV. Muscarinic activation did not directly evoke a [Ca2+]i increase, but subthreshold and suprathreshold depolarization did. Functional axon mapping revealed local axon branching as well as axon collaterals ascending to layers II and III. During blockade of ionotropic glutamatergic AMPA and NMDA receptors, carbachol depolarized layer V neurons by +7.5 ± 3.4 mV. This direct muscarinic depolarization was associated with a conductance increase of 35 ± 10.3% (+4.3 ± 1.25 nS). Intracellular buffering of [Ca2+]i changes did not block this depolarization, but prolonged action potential duration and reduced adaptation of action potential firing. The muscarinic depolarization was neither blocked by combining intracellular Ca2+-buffering (EGTA or BAPTA) with non-specific Ca2+-channel inhibition by Ni+ (1 m m), nor by Ba2+ (1 m m) nor during inhibition of the h-current by 2 m m Cs+. In whole-cell patch-clamp recording, reversal of the muscarinic current occurred at about −45 mV and −5 mV with complete substitution of intrapipette K+ with Cs+. Thus, muscarinic depolarization of EC layer V neurons appears to be primarily mediated by Ca2+-independent activation of non-specific cation channels that conduct K+ about three times as well as Na+. [ABSTRACT FROM AUTHOR]

Abstract The entorhinal cortex (EC) is a key structure in memory formation, relaying sensory information to the hippocampal formation and processed information to the neocortex. EC neurons in the deep layers modulate the transfer of sensory information by the superficial layers and the dentate gyrus, and form the output to the neocortex. Here we characterize two types of EC layer V neurons by their fluorescence morphology, electrophysiology and intracellular Ca2+ signalling using intracellular recording and Ca2+ imaging. Pyramidal neurons show, in response to depolarizing current pulses, regular firing with strong adaptation and a fast and medium afterhyperpolarization (AHP) which are separated by a depolarizing notch and, with hyperpolarizing current injection, a transient sag. Multipolar cells respond to depolarization with delayed firing with very weak adaptation and have no depolarizing notch between fast and medium AHP and no sag with hyperpolarization. The delayed firing was blocked by 30 µm 4-aminopyridine, indicating mediation by the D-type potassium current. Subthreshold depolarization evoked membrane potential oscillations of 2–5 Hz in both cell types and an increase in [Ca2+ ]i of 37 nm in pyramidal and 59 nm in multipolar neurons. Repetitive firing at 10 Hz for 30 s increased [Ca2+ ]i in pyramidal and multipolar neurons by 194 and 295 nm, respectively. Differential temporal firing and Ca2+ signalling suggest specific information processing and synaptic memory storage possibilities in these two layer V cell types of the EC. [ABSTRACT FROM AUTHOR]

Abstract The entorhinal cortex (EC) is a major gateway for sensory information into the hippocampus and receives a cholinergic input from the forebrain. Therefore, we studied muscarinic effects on excitability and intracellular Ca2+ signalling in layer II stellate and layer III pyramidal projection neurons of the EC. In both classes of neurons, local pressure-pulse application of carbachol (1 mm) caused small, atropine-sensitive membrane depolarizations that were not accompanied by any detectable changes in [Ca2+]i. At a higher concentration (10 mm), carbachol induced a larger membrane depolarization associated with synaptic oscillations and epileptiform activity in both classes of neurons. In contrast to the intrinsic theta rhythm in stellate cells with one dominant peak frequency at ∼ 7 Hz, the synaptically mediated oscillation induced by carbachol showed three characteristic peaks in the theta and gamma frequency range at ∼ 11, 23 and 40 Hz. Although carbachol-induced epileptiform activity was associated with increases in intracellular free Ca2+ in both layer II and III cells, the observed [Ca2+]i accumulation was significantly larger in layer III than in layer II cells. Responses to intracellular current injections showed differences in Ca2+ accumulation in layer II and III cells at the same membrane potentials, suggesting a dominant expression of low- and high-voltage-activated Ca2+ channels in these layer II and III cells, respectively. In conclusion, we present evidence for significant differences in the [Ca2+]i regulation between layer II stellate and layer III pyramidal cells of the medial EC. [ABSTRACT FROM AUTHOR]