1. The repolarization of the action potential, and a fast after-hyperpolarization (a.h.p.) were studied in CA1 pyramidal cells (n = 76) in rat hippocampal slices (28-37 degrees C). Single spikes were elicited by brief (1-3 ms) current pulses, at membrane potentials close to rest (-60 to -70 mV). 2. Each action potential was followed by four after-potentials: (a) the fast a.h.p., lasting 2-5 ms; (b) an after-depolarization; (c) a medium a.h.p., (50-100 ms); and (d) a slow a.h.p. (1-2 s). Both the fast a.h.p. and the slow a.h.p. (but not the medium a.h.p.) were inhibited by Ca2+-free medium or Ca2+-channel blockers (Co2+, Mn2+ or Cd2+); but tetraethylammonium (TEA; 0.5-2 nM) blocked only the fast a.h.p., and noradrenaline (2-5 microM) only the slow a.h.p. This suggests that two Ca2+-activated K+ currents were involved: a fast, TEA-sensitive one (IC) underlying the fast a.h.p., and a slow noradrenaline-sensitive one (IAHP) underlying the slow a.h.p. 3. Like the fast a.h.p., spike repolarization seems to depend on a Ca2+-dependent K+ current of the fast, TEA-sensitive kind (IC). The repolarization was slowed by Ca2+-free medium, Co2+, Mn2+, Cd2+, or TEA, but not by noradrenaline. Charybdotoxin (CTX; 30 nM), a scorpion toxin which blocks the large-conductance Ca2+-activated K+ channel in muscle, had a similar effect to TEA. The effects of TEA and Cd2+ (or Mn2+) showed mutual occlusion. Raising the external K+ concentration reduced the fast a.h.p. and slowed the spike repolarization, whereas Cl- loading of the cell was ineffective. 4. The transient K+ current, IA, seems also to contribute to spike repolarization, because: (a) 4-aminopyridine (4-AP; 0.1 mM), which blocks IA, slowed the spike repolarization; (b) depolarizing pre-pulses, which inactivate IA, had a similar effect; (c) hyperpolarizing pre-pulses speeded up the spike repolarization; (d) the effects of 4-AP and pre-pulses persisted during Ca2+ blockade (like IA); and (e) depolarizing pre-pulses reduced the effect of 4-AP. 5. Pre-pulses or 4-AP broadened the spike less, and in a different manner, than Ca2+-free medium, Cd2+, Co2+, Mn2+, TEA or CTX. The former broadening was uniform, with little effect on the fast a.h.p., whereas the latter affected mostly the last two-thirds of the spike repolarization and abolished the fast a.h.p.(ABSTRACT TRUNCATED AT 400 WORDS)Read more
Cloning and sequencing of cDNAs isolated from a rat cortex cDNA library reveals that a gene family encodes several highly homologous K+ channel forming (RCK) proteins. Functional characterization of the channels expressed in Xenopus laevis oocytes following microinjection of in vitro transcribed RCK-specific RNAs shows that each of the RCK proteins forms K+ channels that differ greatly in both their functional and pharmacological properties. This suggests that the molecular basis for the diversity of voltage-gated K+ channels in mammalian brain is based, at least partly, on the expression of several RCK proteins by a family of genes and their assembly to homooligomeric K+ channels with different functional properties.Read more
Hippocampal activity influences neurogenesis in the adult dentate gyrus; however, little is known about the involvement of the hippocampal circuitry in this process. In the subgranular zone of the adult dentate gyrus, neurogenesis involves a series of differentiation steps from radial glia-like stem/progenitor (type-1) cells, to transiently amplifying neuronal progenitor (type-2) cells, to postmitotic neurons. In this study, we conducted GFP-targeted recordings of progenitor cells in fresh hippocampal slices from nestin-GFP mice and found that neuronal progenitor (type-2) cells receive active direct neural inputs from the hippocampal circuitry. This input was GABAergic but not glutamatergic. The GABAergic inputs depolarized type-2 cells because of their elevated [Cl(-)](i). This excitation initiated an increase of [Ca(2+)](i) and the expression of NeuroD. A BrdU-pulse labeling study with GABA(A)-R agonists demonstrated the promotion of neuronal differentiation via this GABAergic excitation. Thus, it appears that GABAergic inputs to hippocampal progenitor cells promote activity-dependent neuronal differentiation.Read more
This study probes the cellular basis for the T wave under baseline and long-QT (LQT) conditions using an arterially perfused canine left ventricular (LV) wedge preparation, which permits direct temporal correlation of cellular transmembrane and ECG events.
Floating microelectrodes were used to record transmembrane action potentials (APs) simultaneously from epicardial, M-region, and endocardial sites or subendocardial Purkinje fibers. A transmural ECG was recorded concurrently. Under baseline and LQT conditions, repolarization of the epicardial action potential, the earliest to repolarize, coincided with the peak of the T wave; repolarization of the M cells, the last to repolarize, coincided with the end of the T wave. Thus, the action potential duration (APD) of the longest M cells determine the QT interval and the Tpeak-Tend interval serves as an index of transmural dispersion of repolarization. Repolarization of Purkinje fibers outlasted that of the M cell but failed to register on the ECG. The morphology of the T wave appeared to be due to currents flowing down voltage gradients on either side of the M region during phase 2 and phase 3 of the ventricular action potential. The interplay between these opposing forces determined the height of the T wave as well as the degree to which the ascending or descending limb of the T wave was interrupted, giving rise to bifurcated T waves and "apparent T-U complexes" under LQT conditions. Spontaneous and stimulation-induced polymorphic ventricular tachycardia with characteristics of torsade de pointes (TdP) developed in the presence of dl-sotalol.
Our results provide the first direct evidence that opposing voltage gradients between epicardium and the M region and endocardium and the M region contribute prominently to the inscription of the ECG T wave under normal conditions and to the widened or bifurcated T wave and long-QT interval observed under LQT conditions. Our data suggest that the "pathophysiological U" wave observed in acquired or congenital LQTS is more likely to be a second component of an interrupted T wave, and argue for use of the term T2 in place of U to describe this event.Read more
We describe an electrophysiological preparation of the neuromuscular junction of the nematode C. elegans, which adds to its considerable genetic and genomic resources. Mutant analysis, pharmacology and patch-clamp recording showed that the body wall muscles of wild-type animals expressed a GABA receptor and two acetylcholine receptors. The muscle GABA response was abolished in animals lacking the GABA receptor gene unc-49. One acetylcholine receptor was activated by the nematocide levamisole. This response was eliminated in mutants lacking either the unc-38 or unc-29 genes, which encode alpha and non-alpha acetylcholine receptor subunits, respectively. The second, previously undescribed, acetylcholine receptor was activated by nicotine, desensitized rapidly and was selectively blocked by dihydro-beta-erythroidine, thus explaining the residual motility of unc-38 and unc-29 mutants. By recording spontaneous endogenous currents and selectively eliminating each of these receptors, we demonstrated that all three receptor types function at neuromuscular synapses.Read more
A long-standing question in neurobiology is whether astrocytes respond to the neuronal release of neurotransmitters in vivo. To address this question, acutely isolated hippocampal slices were loaded with the calcium-sensitive dye Calcium Green-1 and the responses of the astrocytes to electrical stimulation of the Schaffer collaterals were monitored by confocal microscopy. To confirm that the responsive cells were astrocytes, the slices were immunostained for the astrocytic marker glial fibrillary acidic protein. Stimulation of the Schaffer collaterals (50 Hz, 2 sec) resulted in increases in the concentration of intracellular calcium ([Ca2+]i) in the astrocytes located in the stratum radiatum of CA1. The astrocytic responses were blocked by the sodium channel blocker tetrodotoxin, the voltage-dependent calcium channel blocker omega-conotoxin-MVIIC, and the selective metabotropic glutamate receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG). These results suggest that the astrocytic responses were induced by stimulation of metabotropic glutamate receptors on the astrocytes by neuronally released glutamate. The astrocytic responses to neuronal stimulation were enhanced in the presence of the K+ channel antagonist 4-aminopyridine (4-AP). Inhibition of the astrocytic responses in the presence of 4-AP required the presence of both MCPG and the ionotropic glutamate receptor antagonist kynurenic acid. These results suggest that higher levels of neuronal activity result in stimulation of both metabotropic and ionotropic glutamate receptors on the astrocytes. Overall, the results indicate that hippocampal astrocytes in situ are able to respond to the neuronal release of the neurotransmitter glutamate with increases in [Ca2+]i.Read more
Action potentials are binary signals that transmit information via their rate and temporal pattern. In this context, the axon is thought of as a transmission line, devoid of a role in neuronal computation. Here, we show a highly localized role of axonal Kv1 potassium channels in shaping the action potential waveform in the axon initial segment (AIS) of layer 5 pyramidal neurons independent of the soma. Cell-attached recordings revealed a 10-fold increase in Kv1 channel density over the first 50 microm of the AIS. Inactivation of AIS and proximal axonal Kv1 channels, as occurs during slow subthreshold somatodendritic depolarizations, led to a distance-dependent broadening of axonal action potentials, as well as an increase in synaptic strength at proximal axonal terminals. Thus, Kv1 channels are strategically positioned to integrate slow subthreshold signals, providing control of the presynaptic action potential waveform and synaptic coupling in local cortical circuits.Read more
Inherited channelopathies are at the origin of many neurological disorders. Here we report a form of channelopathy that is acquired in experimental temporal lobe epilepsy (TLE), the most common form of epilepsy in adults. The excitability of CA1 pyramidal neuron dendrites was increased in TLE because of decreased availability of A-type potassium ion channels due to transcriptional (loss of channels) and posttranslational (increased channel phosphorylation by extracellular signal-regulated kinase) mechanisms. Kinase inhibition partly reversed dendritic excitability to control levels. Such acquired channelopathy is likely to amplify neuronal activity and may contribute to the initiation and/or propagation of seizures in TLE.Read more
Large-conductance Ca(2+)-activated K(+) channels (BK, also called Maxi-K or Slo channels) are widespread in the vertebrate nervous system, but their functional roles in synaptic transmission in the mammalian brain are largely unknown. By combining electrophysiology and immunogold cytochemistry, we demonstrate the existence of functional BK channels in presynaptic terminals in the hippocampus and compare their functional roles in somata and terminals of CA3 pyramidal cells. Double-labeling immunogold analysis with BK channel and glutamate receptor antibodies indicated that BK channels are targeted to the presynaptic membrane facing the synaptic cleft in terminals of Schaffer collaterals in stratum radiatum. Whole-cell, intracellular, and field-potential recordings from CA1 pyramidal cells showed that the presynaptic BK channels are activated by calcium influx and can contribute to repolarization of the presynaptic action potential (AP) and negative feedback control of Ca(2+) influx and transmitter release. This was observed in the presence of 4-aminopyridine (4-AP, 40-100 microm), which broadened the presynaptic compound action potential. In contrast, the presynaptic BK channels did not contribute significantly to regulation of action potentials or transmitter release under basal experimental conditions, i.e., without 4-AP, even at high stimulation frequencies. This is unlike the situation in the parent cell bodies (CA3 pyramidal cells), where BK channels contribute strongly to action potential repolarization. These results indicate that the functional role of BK channels depends on their subcellular localization.Read more
The hypothalamic arcuate nucleus (ARC) integrates and responds to satiety and hunger signals and forms the origins of the central neural response to perturbations in energy balance. Here we show that rat ARC neurons containing neuropeptide Y (NPY) and agouti-related protein (AgRP), which are conditional pacemakers, are activated by orexigens and inhibited by the anorexigen leptin. We propose a neuron-specific signaling mechanism through which central and peripheral signals engage the central neural anabolic drive.Read more
1. In rabbit and human hearts there are significant differences in the action potential configuration in atrium and ventricle, and the action potential waveform exhibits marked frequency dependence in both tissues. To study the ionic mechanism(s) of these phenomena, the size and time course of the potassium (K+) currents responsible for repolarization have been recorded from single cells using a whole-cell microelectrode voltage clamp method. 2. At physiological heart rates, the action potential in atrial cells has a short plateau phase; however, the rapid early repolarization is strongly frequency dependent. Ventricular myocytes have a long plateau (400-700 ms at 23 degrees C), and the late repolarizing phase of the action potential is much faster in ventricle than in atrium. 3. In both cell types, four different outward currents can be recorded: (i) a large transient outward current, It; (ii) IK(Ca), a smaller Ca2+-dependent K+ current; (iii) IK, a small, maintained time- and voltage-dependent delayed rectifier K+ current; (iv) IK1, an inwardly rectifying K+ current. 4. It, which is responsible for early repolarization, is much larger in atrium than in ventricle. It has very rapid activation and inactivation kinetics but a very slow time course of recovery from inactivation (tau = 5.4 s at 23 degrees C). Our results show that the reactivation kinetics of It are responsible for the pronounced dependence of the shape of the atrial action potential on stimulus frequency. 5. IK(Ca) is variable from cell to cell and is larger in atrium than in ventricle. In both cell types, IK(Ca) is much smaller than It. 6. The delayed rectifier current, IK, is very small and turns on relatively slowly in both cell types. It is therefore not activated strongly during the relatively short plateau of the atrial action potential. Even in ventricle, it contributes only a small repolarizing current. 7. IK1, the inward rectifier K+ current, is much larger in ventricle than in atrium. The current-voltage relationship for IK1 in ventricle exhibits a negative slope conductance between -50 and 0 mV. IK1 is the outward current which generates the resting membrane potential and it modulates the final repolarization phase of the action potential in both cell types. 8. These data strongly suggest that the action potential configuration and its frequency dependence in rabbit atrial and ventricular cells are mainly due to the differences in sizes and kinetics of It and IK1.Read more
A class of K channels in cardiac muscle is reversibly blocked by intracellular adenosine 5'-triphosphate (ATP). The characteristics of this K channel were studied by recording single-channel currents in ventricular cells isolated enzymatically from guinea-pig heart. The reversal potential of single-channel currents agreed well with the K equilibrium potential. Blockers of other K channels, such as tetraethylammonium and 4-aminopyridine, decreased the mean open time of the channel. The chord conductance increased as the 0.24th power of the K concentration on the outer surface of the membrane, and showed a marked inward-going rectification on strong depolarizations. The degree of rectification was larger with increasing Na concentration on the inner side of the membrane. The kinetics of the channel were almost voltage independent, but depended on the concentration of intracellular ATP. The conductance of the channel was not affected by ATP. When channel kinetics were examined in the presence of ATP, the distribution of open times and closed times was fitted well with a sum of two exponential components. When ATP concentration was increased, the time constants obtained from the open-time histogram decreased and those from the closed-time histogram increased, resulting in a decrease of the open-state probability. The channel was blocked by ATP, adenosine 5'-diphosphate,5'-adenylylimidodiphosphate, guanosine 5'-triphosphate and uridine 5'-triphosphate, but not by adenosine 5'-monophosphate, creatine phosphate, creatine or adenosine. Plots of the open-state probability versus the ATP concentration revealed Michaelis-Menten saturation kinetics with strong co-operativity of multiple receptor sites (Hill coefficient 3-4, concentration of half-saturation 0.5 mM). It was concluded that this K channel has three or four receptor sites selective for triphosphate nucleotide on the inner surface of the membrane, and that the channel is blocked through the binding of agonists to the receptors.Read more
Depolarization of human atrial myocytes activates a transient outward current that rapidly inactivates, leaving a sustained outward current after continued depolarization. To evaluate the ionic mechanism underlying this sustained current (Isus), we applied whole-cell voltage-clamp techniques to single myocytes isolated from right atrial specimens obtained from patients undergoing coronary bypass surgery. The magnitude of Isus was constant for up to 10 seconds at +30 mV and was unaffected by 40 mmol/L tetraethylammonium, 100 nmol/L dendrotoxin, 1 mmol/L Ba2+, 0.1 mumol/L atropine, or removal of Cl- in the superfusate. Isus could be distinguished from the 4-aminopyridine (4AP)-sensitive transient outward current (Ito1) by differences in voltage-dependent inactivation (1000-millisecond prepulse to -20 mV reduced Ito1 by 91.7 +/- 0.1% [mean +/- SEM], P < .001, versus 9.4 +/- 0.4% reduction of Isus) and 4AP sensitivity (IC50 for block of Ito1, 1.96 mmol/L; for Isus, 49 mumol/L). Isus activation had a voltage threshold near -30 mV, a half-activation voltage of -4.3 mV, and a slope factor of 8.0 mV. Isus was not inactivated by 1000-millisecond prepulses but was reduced by 16 +/- 8% (P < .05) at a holding potential of -20 mV relative to values at a holding potential of -80 mV. Isus activated very rapidly, with time constants (tau) at 25 degrees C ranging from 18.2 +/- 1.8 to 2.1 +/- 0.2 milliseconds at -10 to +50 mV, two orders of magnitude faster than previously described kinetics of the rapid component of the delayed rectifier K+ current. At 16 degrees C, Isus activation was greatly slowed (tau at +10 mV, 46.7 +/- 4.1 milliseconds; tau at 25 degrees C, 7.1 +/- 0.8 milliseconds; P < .01), and the envelope of tails test was satisfied. The reversal potential of Isus tail currents changed linearly with log [K+]o (slope, 55.3 +/- 2.9 mV per decade), and the fully activated current-voltage relation showed substantial outward rectification. Selective inhibition of Isus with 50 mumol/L 4AP increased human atrial action potential duration by 66 +/- 11% (P < .01). In conclusion, Isus in human atrial myocytes is due to a very rapidly activating delayed rectifier K+ current, which shows limited slow inactivation, is insensitive to tetraethylammonium, Ba2+, and dendrotoxin, and is highly sensitive to 4AP. These properties resemble the characteristics of channels encoded by the Kv1.5 group of cardiac cDNAs and may represent a physiologically significant manifestation of such channels in human atrium.Read more
A single mossy fiber input contains several release sites and is located on the proximal portion of the apical dendrite of CA3 neurons. It is, therefore, well suited to exert a strong influence on pyramidal cell excitability. Accordingly, the mossy fiber synapse has been referred to as a detonator or teacher synapse in autoassociative network models of the hippocampus. The very low firing rates of granule cells [Jung, M. W. & McNaughton, B. L. (1993) Hippocampus 3, 165-182], which give rise to the mossy fibers, raise the question of how the mossy fiber synapse temporally integrates synaptic activity. We have therefore addressed the frequency dependence of mossy fiber transmission and compared it to associational/commissural synapses in the CA3 region of the hippocampus. Paired pulse facilitation had a similar time course, but was 2-fold greater for mossy fiber synapses. Frequency facilitation, during which repetitive stimulation causes a reversible growth in synaptic transmission, was markedly different at the two synapses. At associational/ commissural synapses facilitation occurred only at frequencies greater than once every 10 s and reached a magnitude of about 125% of control. At mossy fiber synapses, facilitation occurred at frequencies as low as once every 40 s and reached a magnitude of 6-fold. Frequency facilitation was dependent on a rise in intraterminal Ca2+ and activation of Ca2+/calmodulin-dependent kinase II, and was greatly reduced at synapses expressing mossy fiber long-term potentiation. These results indicate that the mossy fiber synapse is able to integrate granule cell spiking activity over a broad range of frequencies, and this dynamic range is substantially reduced by long-term potentiation.Read more
Seizures in patients presenting with mesial temporal lobe epilepsy result from the interaction among neuronal networks in limbic structures such as the hippocampus, amygdala and entorhinal cortex. Mesial temporal lobe epilepsy, one of the most common forms of partial epilepsy in adulthood, is generally accompanied by a pattern of brain damage known as mesial temporal sclerosis. Limbic seizures can be mimicked in vitro using preparations of combined hippocampus-entorhinal cortex slices perfused with artificial cerebrospinal fluid containing convulsants or nominally zero Mg(2+), in order to produce epileptiform synchronization. Here, we summarize experimental evidence obtained in such slices from rodents. These data indicate that in control animals: (i) prolonged, NMDA receptor-dependent epileptiform discharges, resembling electrographic limbic seizures, originate in the entorhinal cortex from where they propagate to the hippocampus via the perforant path-dentate gyrus route; (ii) the initiation and maintenance of these ictal discharges is paradoxically contributed by GABA (mainly type A) receptor-mediated mechanisms; and (iii) CA3 outputs, which relay a continuous pattern of interictal discharge at approximately 1Hz, control rather than sustain ictal discharge generation in entorhinal cortex. Recent work indicates that such a control is weakened in the pilocarpine model of epilepsy (presumably as a result of CA3 cell damage). In addition, in these experiments electrographic seizure activity spreads directly to the CA1-subiculum regions through the temporoammonic pathway. Studies reviewed here indicate that these changes in network interactions, along with other mechanisms of synaptic plasticity (e.g. axonal sprouting, decreased activation of interneurons, upregulation of bursting neurons) can confer to the epileptic, damaged limbic system, the ability to produce recurrent limbic seizures as seen in patients with mesial temporal lobe epilepsy.Read more
Computational biology is a powerful tool for elucidating arrhythmogenic mechanisms at the cellular level, where complex interactions between ionic processes determine behavior. A novel theoretical model of the canine ventricular epicardial action potential and calcium cycling was developed and used to investigate ionic mechanisms underlying Ca2+ transient (CaT) and action potential duration (APD) rate dependence.
The Ca2+/calmodulin-dependent protein kinase (CaMKII) regulatory pathway was integrated into the model, which included a novel Ca2+-release formulation, Ca2+ subspace, dynamic chloride handling, and formulations for major ion currents based on canine ventricular data. Decreasing pacing cycle length from 8000 to 300 ms shortened APD primarily because of I(Ca(L)) reduction, with additional contributions from I(to1), I(NaK), and late I(Na). CaT amplitude increased as cycle length decreased from 8000 to 500 ms. This positive rate-dependent property depended on CaMKII activity.
CaMKII is an important determinant of the rate dependence of CaT but not of APD, which depends on ion-channel kinetics. The model of CaMKII regulation may serve as a paradigm for modeling effects of other regulatory pathways on cell function.Read more
Focal activation of glutamate receptors in distal dendrites of hippocampal pyramidal cells triggers voltage-dependent Ca(2+) channel-mediated plateau potentials that are confined to the stimulated dendrite. We examined the role of dendritic K(+) conductances in determining the amplitude, duration, and spatial compartmentalization of plateau potentials. Manipulations that blocked SK-type Ca(2+)-activated K(+) channels, including apamin and BAPTA dialysis, increased the duration of plateau potentials without affecting their amplitude or compartmentalization. Manipulations that blocked Kv4.2 A-type K(+) channels, including a dominant-negative Kv4.2 construct and 4-aminopyridine, increased the amplitude of plateau potentials by allowing them to recruit neighboring dendrites. Prolongation of plateau potentials or block of Kv4.2 channels at branch points facilitated the ability of dendritic excitation to trigger fast action potentials. SK channels thus underlie repolarization of dendritic plateau potentials, whereas Kv4.2 channels confine these potentials to single dendritic branches, and both act in concert to regulate synaptic integration.Read more
Long-term potentiation (LTP) is currently the best available cellular model for learning and memory in the mammalian brain. In the CA1 region of the hippocampus, as well as in many other areas of the CNS, its induction requires a rise in postsynaptic Ca2+ via activation of NMDA receptors. What happens after the rise in postsynaptic Ca2+ is less clear. This paper summarizes experiments performed over the last decade in slice preparations that address the site of expression of LTP. While a large number of laboratories have contributed importantly to this issue, this review will rely primarily on experiments performed in the authors' laboratory. The experiments to be discussed can be broadly divided into two groups: those designed to determine if an increase in glutamate release occurs during LTP and those designed to determine if a change in postsynaptic sensitivity to glutamate occurs during LTP. Experiments in the first category include the analysis of dual-component excitatory postsynaptic currents (EPSCs), paired-pulse facilitation, saturating release probability, the use of MK-801 to measure release probability, and glial glutamate transporter currents to measure directly the synaptic release of glutamate. Experiments in the second category include analysis of miniature EPSC amplitudes, measurements of synaptic potency, the consequences of loading cells with the constitutively activated form of CaM kinase II, and the evidence that during LTP postsynaptically silent synapses become functional. We will argue that, while numerous experiments fail to support a presynaptic expression mechanism, many experiments do point to a postsynaptic expression mechanism. The decrease in synaptic failures during LTP, the only generally accepted experimental result that supports a presynaptic expression mechanism, can be explained by postsynaptically silent synapses. Future directions for research in this field include activity-dependent targeting of glutamate receptors and the functional consequences of phosphorylation of AMPA receptors.Read more
The synaptic control of the astrocytic intracellular Ca2+ is crucial in the reciprocal astrocyte-neuron communication. Using electrophysiological and Ca2+ imaging techniques in rat hippocampal slices, we investigated the astrocytic Ca2+ signal modulation induced by synaptic terminals that use glutamate and acetylcholine. Ca2+ elevations were evoked by glutamate released from Schaffer collaterals and by acetylcholine, but not glutamate, released by alveus stimulation, indicating that astrocytes discriminate the activity of different synapses belonging to different axon pathways. The Ca2+ signal was modulated bidirectionally by simultaneous activation of both pathways, being depressed at high stimulation frequencies and enhanced at low frequencies. The Ca2+ modulation was attributable to astrocytic intrinsic properties, occurred at discrete regions of the processes, and controlled the intracellular expansion of the Ca2+ signal. In turn, astrocyte Ca2+ signal elicited NMDA receptor-mediated currents in pyramidal neurons. Therefore, because astrocytes discriminate and integrate synaptic information, we propose that they can be considered as cellular elements involved in the information processing by the nervous system.Read more
The properties of bladder afferent neurons in L6 and S1 dorsal root ganglia of adult rats were evaluated after chronic bladder inflammation induced by 2 week treatment with cyclophosphamide (CYP; 75 mg/kg). Whole-cell patch-clamp recordings revealed that most (70%) of the dissociated bladder afferent neurons from control rats were capsaicin sensitive, with high-threshold long-duration action potentials that were not blocked by tetrodotoxin (TTX; 1 microM). These neurons exhibited membrane potential relaxations during voltage responses elicited by depolarizing current pulses and phasic firing during sustained membrane depolarization. After CYP treatment, a similar proportion (71%) of bladder afferent neurons were capsaicin sensitive with TTX-resistant spikes. However, the neurons were significantly larger in size (diameter 29.6 +/- 1.0 micrometer vs 23.6 +/- 0.8 micrometer in controls). TTX-resistant bladder afferent neurons from CYP-treated rats exhibited lower thresholds for spike activation (-25.4 +/- 0.5 mV) than those from control rats (-21.4 +/- 0.9 mV) and did not exhibit membrane potential relaxation during depolarization. Seventy percent of TTX-resistant bladder afferent neurons from CYP-treated rats exhibited tonic firing (average 12.3 +/- 1.4 spikes during a 500 msec depolarizing pulse) versus phasic firing (1.2 +/- 0.2 spikes) in normal bladder afferent neurons. Application of 4-aminopyridine (1 mM) to normal TTX-resistant bladder afferent neurons mimicked the changes in firing properties after CYP treatment. The peak density of an A-type K+ current (IA) during depolarizations to 0 mV in TTX-resistant bladder afferent neurons from CYP-treated rats was significantly smaller (42.9 pA/pF) than that from control rats (109.4 pA/pF), and the inactivation curve of the IA current was displaced to more hyperpolarized levels by approximately 15 mV after CYP treatment. These data suggest that chronic inflammation induces somal hypertrophy and increases the excitability of C-fiber bladder afferent neurons by suppressing IA channels. Similar electrical changes in sensory pathways may contribute to cystitis-induced pain and hyperactivity of the bladder.Read more
Developmental changes in electrophysiological membrane properties of mouse cochlear inner hair cells (IHCs) were studied from just after terminal differentiation up to functional maturity. As early as embryonic day 14.5 (E14.5) newly differentiated IHCs express a very small outward K+ current that is largely insensitive to 4-aminopyridine (4-AP). One day later the inward rectifier, IK1, is first observed. These immature cells initially exhibit only slow graded voltage responses under current clamp. From E17.5 spontaneous action potentials occur. During the first week of postnatal development, the outward K+ current steadily increases in size and a progressively larger fraction of the current is sensitive to 4-AP. During the second postnatal week, the activation of the 4-AP-sensitive current, by now contributing about half of the outward K+ current, shifts in the hyperpolarizing direction. Together with an increase in size of IK1, this hyperpolarizes the cell, thus inhibiting the spontaneous spike activity, although spikes could still be evoked upon depolarizing current injection. Starting at about the onset of hearing (postnatal day 12, P12) immature IHCs make the final steps towards fully functional sensory receptors with fast graded voltage responses. This is achieved mainly by the expression of the large-conductance Ca2+-activated K+ current IK,f, but also of a current indistinguishable from the negatively activating IK,n previously described in mature outer hair cells (OHCs). The 4-AP-sensitive current continues to increase after the onset of hearing to form the major part of the mature delayed rectifier, IK,s. By P20 IHCs appear mature in terms of their complement of K+ conductances.Read more
Clinical studies suggested that fampridine (4-aminopyridine) improves motor function in people with multiple sclerosis. This phase III study assessed efficacy and safety of oral, sustained-release fampridine in people with ambulatory deficits due to multiple sclerosis.
We undertook a randomised, multicentre, double-blind, controlled phase III trial. We randomly assigned 301 patients with any type of multiple sclerosis to 14 weeks of treatment with either fampridine (10 mg twice daily; n=229) or placebo (n=72), using a computer-generated sequence stratified by centre. We used consistent improvement on timed 25-foot walk to define response, with proportion of timed walk responders in each treatment group as the primary outcome. We used the 12-item multiple sclerosis walking scale to validate the clinical significance of the response criterion. Efficacy analyses were based on a modified intention-to-treat population (n=296), which included all patients with any post-treatment efficacy data. The study is registered with ClinicalTrials.gov, number NCT00127530.
The proportion of timed walk responders was higher in the fampridine group (78/224 or 35%) than in the placebo group (6/72 or 8%; p<0.0001). Improvement in walking speed in fampridine-treated timed walk responders, which was maintained throughout the treatment period, was 25.2% (95% CI 21.5% to 28.8%) and 4.7% (1.0% to 8.4%) in the placebo group. Timed walk responders showed greater improvement in 12-item multiple sclerosis walking scale scores (-6.84, 95% CI -9.65 to -4.02) than timed walk non-responders (0.05, -1.48 to 1.57; p=0.0002). Safety data were consistent with previous studies.
Fampridine improved walking ability in some people with multiple sclerosis. This improvement was associated with a reduction of patients' reported ambulatory disability, and is a clinically meaningful therapeutic benefit.Read more
The calcium channel blockers, verapamil and diltiazem, inhibit phytohemagglutinin (PHA)-induced mitogenesis at concentrations that block the T lymphocyte K channel currents. K channel blockers also inhibit the allogeneic mixed lymphocyte response in a dose-dependent manner with the same potency sequence as for block of K currents. K channel blockers inhibit PHA-stimulated mitogenesis only if added during the first 20-30 h after PHA addition, but not later, indicating a requirement for functional K channels during this period. We investigated the effect of K channel blockers on various aspects of protein synthesis for two reasons: first, protein synthesis appears to be necessary for the events leading to DNA synthesis, and second, the increase in the protein synthetic rate commences during the first 24-48 h after PHA addition. PHA-induced total protein synthesis was reduced to the level in unstimulated T lymphocytes by K channel blockers in a dose-dependent manner with the same potency sequence as for the block of K currents and inhibition of [3H]thymidine incorporation. Two-dimensional gel electrophoresis demonstrated that although the synthesis of the majority of proteins was reduced by K channel blockers to the level in unstimulated T cells, some proteins continued to be synthesized at an enhanced rate compared with resting cells. Two proteins, S and T, detected by two-dimensional gel electrophoresis in unstimulated T lymphocytes, appeared to be reduced in intensity in gels of PHA-treated T lymphocytes, in contrast to the increased synthesis of the remaining proteins. 4-Aminopyridine (4-AP), at concentrations that inhibit protein synthesis, prevented the apparent PHA-induced reduction of proteins S and T. These proteins may play a role in maintaining the T lymphocyte in a resting state and may be related to the translation inhibitory factors reported to be present at a higher specific activity in quiescent T lymphocytes than in PHA-activated T cells. The expression of the IL-2 receptor (Tac) during T lymphocyte activation was not altered by K channel blockers, whereas the production of interleukin 2 (IL-2) was reduced to the level in unstimulated T lymphocytes. Exogenous IL-2 partially relieved the inhibition of mitogenesis by low, but not by high, concentrations of 4-AP. These experiments clarify the role of K channels in T lymphocyte activation and suggest that functional K channels are required either for protein synthesis or for events leading to protein synthesis.Read more