Decreased Burst Duration Research Articles

Abnormal beta bursts of depression in the orbitofrontal cortex and its relationship with clinical symptoms

BackgroundRecent researches have reported that frequency-specific patterns of neural activity contain not only rhythmically sustained oscillations but also transient-bursts of isolated events. The aim of this study was to investigated the correlation between beta burst and depression in order to explore depressive disease and the neurological underpinnings of disease-related symptoms. MethodsWe collected resting-state MEG recordings from 30 depressive patients and a matched 40 healthy controls. A Hidden Markov Model (HMM) was applied on source-space time courses for 78 cortical regions of the AAL atlas and the temporal characteristics of beta burst from the matched HMM states were captured. Group differences were evaluated on these beta burst characteristics after permutation tests and, for the depressive group, associations between burst characteristics and clinical symptom severity were determined using Spearman correlation coefficients. ResultsAt a threshold of p=0.05corrected, burst characteristics revealed significant differences between depression patients and controls at the group level, including increased burst amplitude in frontal lobe, decreased burst duration in occipital regions, increased burst rate and decreased burst interval time in some brain regions. Furthermore, burst amplitude in the orbitofrontal cortex (OFC) was positively related to the severity of sleep disturbance and burst rate in the OFC was negatively related to the severity of anxiety in depression patients. ConclusionsThe findings highlight OFC may be a targeted area responsible for the anxiety and sleep disturbance symptom by abnormal beta burst in depressive patients and beta burst characteristics of OFC might serve as a neuro-marker for the depression.

Membrane excitabilities in neonatal rat mesencephalic trigeminal neurons under dietary zinc deficiency.

The present study aimed to evaluate the effects of zinc deprivation on the properties of membrane and spike-discharge features of mesencephalic trigeminal neurons (MTNs), which are important sensory neurons for oral-motor reflexes and rhythmical jaw movements. Neonatal Sprague Dawley rats (P10-12) were distributed equally into a normal diet group and a zinc-deficient diet (ZD) group. Whole cell patch-clamp recordings were obtained from MTNs from coronal brain slices. Passive membrane properties showed a modest depolarized membrane potential and decreased cell capacitance in the ZD group. Zinc deprivation decreased the minimal current amplitude, which induced an action potential and increased the amplitude of afterhyperpolarization following the action potential. Negligible changes were observed for other action potential properties. A decreased burst duration was observed, accompanied by hastened spike frequency adaptation in the burst discharge. There was no difference in the resonant properties at both the subthreshold depolarized potential and hyperpolarized membrane potential between the control and ZD groups. These results suggests that neither the persistent sodium conductance nor slow inwardly rectifying conductance were altered; however, there appeared to be an increase in Ca2+-dependent K+ conductance in zincdeficient MTNs.

Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.

Exaggerated basal ganglia beta activity (13–35 Hz) is commonly found in patients with Parkinson’s disease and can be suppressed by dopaminergic medication, with the degree of suppression being correlated with the improvement in motor symptoms. Importantly, beta activity is not continuously elevated, but fluctuates to give beta bursts. The percentage number of longer beta bursts in a given interval is positively correlated with clinical impairment in Parkinson’s disease patients. Here we determine whether the characteristics of beta bursts are dependent on dopaminergic state. Local field potentials were recorded from the subthalamic nucleus of eight Parkinson’s disease patients during temporary lead externalization during surgery for deep brain stimulation. The recordings took place with the patient quietly seated following overnight withdrawal of levodopa and after administration of levodopa. Beta bursts were defined by applying a common amplitude threshold and burst characteristics were compared between the two drug conditions. The amplitude of beta bursts, indicative of the degree of local neural synchronization, progressively increased with burst duration. Treatment with levodopa limited this evolution leading to a relative increase of shorter, lower amplitude bursts. Synchronization, however, was not limited to local neural populations during bursts, but also, when such bursts were cotemporaneous across the hemispheres, was evidenced by bilateral phase synchronization. The probability of beta bursts and the proportion of cotemporaneous bursts were reduced by levodopa. The percentage number of longer beta bursts in a given interval was positively related to motor impairment, while the opposite was true for the percentage number of short duration beta bursts. Importantly, the decrease in burst duration was also correlated with the motor improvement. In conclusion, we demonstrate that long duration beta bursts are associated with an increase in local and interhemispheric synchronization. This may compromise information coding capacity and thereby motor processing. Dopaminergic activity limits this uncontrolled beta synchronization by terminating long duration beta bursts, with positive consequences on network state and motor symptoms.

Unique Configurations of Compression and Truncation of Neuronal Activity Underlie l-DOPA-Induced Selection of Motor Patterns in Aplysia.

A key issue in neuroscience is understanding the ways in which neuromodulators such as dopamine modify neuronal activity to mediate selection of distinct motor patterns. We addressed this issue by applying either low or high concentrations of l-DOPA (40 or 250 μM) and then monitoring activity of up to 130 neurons simultaneously in the feeding circuitry of Aplysia using a voltage-sensitive dye (RH-155). l-DOPA selected one of two distinct buccal motor patterns (BMPs): intermediate (low l-DOPA) or bite (high l-DOPA) patterns. The selection of intermediate BMPs was associated with shortening of the second phase of the BMP (retraction), whereas the selection of bite BMPs was associated with shortening of both phases of the BMP (protraction and retraction). Selection of intermediate BMPs was also associated with truncation of individual neuron spike activity (decreased burst duration but no change in spike frequency or burst latency) in neurons active during retraction. In contrast, selection of bite BMPs was associated with compression of spike activity (decreased burst latency and duration and increased spike frequency) in neurons projecting through specific nerves, as well as increased spike frequency of protraction neurons. Finally, large-scale voltage-sensitive dye recordings delineated the spatial distribution of neurons active during BMPs and the modification of that distribution by the two concentrations of l-DOPA.

Caffeine stimulates locomotor activity in the mammalian spinal cord via adenosine A1 receptor-dopamine D1 receptor interaction and PKA-dependent mechanisms

Caffeine is a potent psychostimulant that can have significant and widely variable effects on the activity of multiple neuronal pathways. The most pronounced caffeine-induced behavioral effect seen in rodents is to increase locomotor activity which has been linked to a dose-dependent inhibition of A1 and A2A receptors. The effects of caffeine at the level of the lumbar spinal central pattern generator (CPG) network for hindlimb locomotion are lacking. We assessed the effects of caffeine to the locomotor function of the spinal CPG network via extracellular ventral root recordings using the isolated neonatal mouse spinal cord preparation. Addition of caffeine and of an A1 receptor antagonist significantly decreased the cycle period accelerating the ongoing locomotor rhythm, while decreasing burst duration reversibly in most preparations suggesting the role of A1 receptors as the primary target of caffeine. Caffeine and an A1 receptor antagonist failed to stimulate ongoing locomotor activity in the absence of dopamine or in the presence of a D1 receptor antagonist supporting A1/D1 receptor-dependent mechanism of action. The use of caffeine or an A1 receptor blocker failed to stimulate an ongoing locomotor rhythm in the presence of a blocker of the cAMP-dependent protein kinase (PKA) supporting the need of this intracellular pathway for the modulatory effects of caffeine to occur. These results support a stimulant effect of caffeine on the lumbar spinal network controlling hindlimb locomotion through the inhibition of A1 receptors and subsequent activation of D1 receptors via a PKA-dependent intracellular mechanism.

Octopamine modulates a central pattern generator associated with egg-laying in the locust, Locusta migratoria

Egg-laying in Locusta migratoria involves the control of a variety of complex behavioural patterns including those that regulate digging of the oviposition hole and retention of eggs during digging. These two behavioural patterns are under the control of central pattern generators (CPGs). The digging and egg-retention CPGs are coordinated and integrated with overlapping locations of neural substrate within the VIIth and VIIIth abdominal ganglia of the central nervous system (CNS). In fact, the egg-retention CPG of the VIIth abdominal ganglion is involved in both egg-retention and protraction of the abdomen during digging. The biogenic amine, octopamine, has peripheral effects on oviduct muscle, relaxing basal tension of the lateral and upper common oviduct and enabling egg passage. Here we show that octopamine also modulates the pattern of the egg-retention CPG by altering the motor pattern that controls the external ventral protractor of the VIIth abdominal segment. There is no change in the motor pattern that goes to the oviducts. Octopamine decreased the frequency of the largest amplitude action potential and decreased burst duration while leading to an increase in cycle duration and interburst interval. The effects of octopamine were greatly reduced in the presence of the α-adrenergic blocker, phentolamine, indicating that the action of octopamine was via a receptor. Thus, octopamine orchestrates events that can lead to oviposition, centrally inhibiting the digging behavior and peripherally relaxing the lateral and common oviducts to enable egg-laying.

Influence of the SCN1A IVS5N + 5 G>A Polymorphism on Therapy with Carbamazepine for Epilepsy

Carbamazepine (CBZ) blocks neuronal sodium channels in a voltage- and frequency-dependent manner, delaying the recovery of the channels from the inactivated state, reducing the number of action potentials within a burst, and decreasing burst duration. The α-subunit of the first neuronal sodium channel (SCN1A) is a major gene in different epilepsies. A synonymous polymorphism (SCN1A IVS5N + 5 G>A or rs3812718) is common in exon 5 of this gene. Mutations in the α-unit of this gene are associated with CBZ-resistant epilepsy and a higher maintenance dose of CBZ. We have investigated the association of this single nucleotide polymorphism (SNP) and epilepsy, efficacy and dose-dependence of CBZ therapy in 147 adult Macedonian patients and 137 non epileptic controls. No significant differences in allelic frequencies and genotype distribution were found between patients and controls (p = 0.94278), or between CBZ-responsive and unresponsive patients (p = 0.55449). An association between the A allele and a higher maintenance dose in CBZ-responsive patients was detected. No statistical difference was found between the plasma levels of CBZ and genotype of patients receiving the same dose, indicating that the variant exerts its effect at the level of receptor responsiveness. The predictive value of pretreatment testing showed a minor insignificant difference between patients with different genotypes, primarily due to a small number of patients.

Studies on Correlated Pairs at the NBD-NBD and NBD-TMD Interfaces in CFTR

CFTR is an ATP-binding cassette (ABC)-protein, which contains two Nucleotide Binding Domains (NBDs) and two Transmembrane Domains (TMDs), but is unique among these proteins in that ATP binding, formation of a tight NBD-dimer (sandwiching two ATPs at two composite sites) and hydrolysis of one nucleotide drives opening/closure of a transmembrane chloride channel pore. We investigated interactions between residues at the NBD1-NBD2 interface of the catalytically inactive site 1, and the NBD-TMD boundary. Target pairs suggested by coevolution analysis were tested experimentally using mutant cycles. At the NBD1-NBD2 interface we studied pairs T460 (NBD1 Walker A)-H1348 (NBD2 signature sequence), and T460-H1375 (NBD2 D-loop). Mutation T460S decreased burst duration (τb) and open probability (Po), mutation H1348A markedly prolonged τb and increased Po. Mutation H1375A lengthened τb while slightly reducing Po. But mutant cycles built on τb, maximal opening rates, as well as Po and closing rates in non-hydrolytic (K1250R) background have so far not revealed significant changes in coupling between either positions 460-1348 or positions 460-1375 during various steps of the gating cycle. Hence we conclude that site 1 does not move markedly during the gating cycle, consistent with only partial NBD-dimer separation after ATP hydrolysis.At the NBD-TMD interface we studied positions M961 (site of CF-mutation in coupling helix 3) and A1256 (NBD2). Although single mutations M961L and A1256V decreased apparent ATP-affinities, these effects were additive, indicating no change in coupling between these positions upon ATP binding to closed channels. Po and τb were unaffected by these mutations. He et al. (JBC 283:26383) showed that L1260 and L1261 are in close contact with M961, and chemical crosslinking of L1261C with M961C lowers Po. Gating of mutants L1260A and L1261A paired with mutation M961L is under investigation.

Task specific adaptations in rat locomotion: Runway versus horizontal ladder

In walking quadrupeds the alternating activity pattern of antagonistic leg muscles and the coordination between legs is orchestrated by central pattern generating networks within the spinal cord. These networks are activated by tonic input from the reticular formation in the brainstem. Under more challenging conditions, such as walking on a horizontal ladder, successful locomotion relies upon additional context dependent input from pathways such as the cortico- and rubro-spinal tracts. In this study we used electromyographic and kinematic approaches to characterize the adaptations in the walking pattern in adult uninjured rats crossing a horizontal ladder. We found that the placement of a hind limb on a rung precisely followed the placement of the ipsilateral fore limb. This is different to normal walking where the hind limb is placed behind the position of the ipsilateral fore limb. The increased reach of the hind limbs is achieved by increased flexion of the hip and rotation of the pelvis during the swing phase. Electromyographic observations showed decreased burst duration in Tibialis anterior an ankle flexor muscle. Further changes in the muscle activity pattern were likely due to the reduced stepping frequency during ladder walking. Following a lesion of the dorsal column, containing major parts of the corticospinal tract, we found an increased number of stepping errors and changes in the stepping strategy. The step length of the fore limbs was reduced and the hind limbs were frequently positioned on rungs other than those occupied by the fore limb.

Effects of anticonvulsants on soman-induced epileptiform activity in the guinea-pig in vitro hippocampus

Seizures arising from acetylcholinesterase inhibition are a feature of organophosphate anticholinesterase intoxication. Although benzodiazepines are effective against these seizures, alternative anticonvulsant drugs may possess greater efficacy and fewer side-effects. We have investigated in the guinea-pig hippocampal slice preparation the ability of a series of anticonvulsants to suppress epileptiform bursting induced by the irreversible organophosphate anticholinesterase, soman (100 nM). Carbamazepine (300 μM), phenytoin (100 μM), topiramate (100–300 μM) and retigabine (1–30 μM) reduced the frequency of bursting but only carbamazepine and phenytoin induced a concurrent reduction in burst duration. Felbamate (100–500 μM) and clomethiazole (100–300 μM) had no effect on burst frequency but decreased burst duration. Clozapine (3–30 μM) reduced the frequency but did not influence burst duration. Levetiracetam (100–300 μM) and gabapentin (100–300 μM) were without effect. These data suggest that several compounds, in particular clomethiazole, clozapine, felbamate, topiramate and retigabine, merit further evaluation as possible treatments for organophosphate poisoning.

The effect of walking aids on muscle activation patterns during walking in stroke patients

The purpose of this study was to investigate changes in muscle activation patterns with respect to timing and amplitude that occur when subjects with stroke walk with and without a walking aid. This knowledge could help therapists in deciding whether or not patients should use a cane or quad stick while walking. Thirteen patients suffering from a first unilateral ischemic stroke participated in the study. Surface electromyography (SEMG) of the erector spinae, gluteus maximus, gluteus medius, vastus lateralis, semitendinosus, gastrocnemius and tibialis anterior of the affected side were measured during three different conditions: (1) walking without a walking aid, (2) walking with a cane and (3) walking with a quad stick. Timing and amplitude parameters of the activation patterns were quantified using an objective burst detection algorithm and statistically evaluated. Results showed a statistically significant and clinically relevant decrease in burst duration of both erector spinae and tibialis anterior when walking with a cane. The amplitude of the vastus lateralis and tibialis anterior dropped when patients walked with a cane and quad stick. The use of a cane should be considered when therapy is given to stroke patients to achieve normal muscle activation patterns.

Influence of hamstring lengthening on muscle activation timing.

The purpose of this study was to describe the changes in muscle activation patterns using surface electromyography (sEMG) during walking in patients with cerebral palsy (CP), before and after hamstring lengthening. In the current clinical use of sEMG during walking in CP for pre-operative planning, various authors have observed that timing of muscle activation patterns hardly changes after surgical intervention. This observation is based on tendon transfer studies and visual interpretation of raw EMG signals. Little is known about the effect of muscle lengthening on muscle activation patterns of the lengthened muscles and their antagonists. Fifteen children with CP comprising a total of 23 hamstring lengthenings were included in this study. Surface EMG of semitendinosus and vastus lateralis was measured before and after surgery. Timing parameters of the sEMG patterns were quantified, using an objective burst detection algorithm and statistically evaluated. Results showed that hamstring lengthening causes statistically significant differences in timing of both the semitendinosus and vastus lateralis. It is concluded that timing parameters of operated muscles and their antagonists after surgery do change. The delayed off-time of the semitendinosus and the decreased burst duration of the antagonist (the vastus lateralis) after surgery were the most important changes.

Effect of lidocaine and NMDA injections into the medial pontobulbar reticular formation on mastication evoked by cortical stimulation in anaesthetized rabbits.

Neurons of the dorsal nucleus reticularis pontis caudalis (nPontc) fire rhythmically during fictive mastication, while neurons of the ventral half tend to fire tonically (Westberg et al., 2001). This paper describes the changes in the pattern of rhythmical mastication elicited by stimulation of the sensorimotor cortex during inhibition or excitation of neurons in this nucleus and adjacent parts of nucleus reticularis gigantocellularis (Rgc) in the anaesthetized rabbit. Masticatory movements and electromyographic (EMG) activity of the masseter and digastric muscles produced by cortical stimulation were recorded before, during and after injections of a local anaesthetic (lidocaine) or excitatory amino acid N-methyl-d-aspartate (NMDA) into nPontc and Rgc through a microsyringe with attached microelectrode to record neuronal activity. Lidocaine inhibited local neurons and modified the motor program, and the effects varied with the site of injection. Most injections into the ventral half of nPontc increased cycle duration, digastric burst duration and burst area. The action of lidocaine in dorsal nPontc was more variable, although burst duration and area were often decreased. The effects on the muscle activity were always bilateral. Lidocaine block of the rostromedial part of Rgc had no effect on movements or on EMGs. Injections of NMDA excited local neurons and when injected into ventral nPontc, it completely blocked mastication. Dorsal injections either had no effect or increased cycle frequency, while decreasing burst duration and area. No increases in EMG burst duration or area were observed with NMDA. Our findings suggest that neurons of ventral nPontc tonically inhibit other parts of the central pattern generator during mastication, while dorsal neurons have mixed effects. We incorporated these findings into a new model of the masticatory central pattern generator.

Role of synaptic inhibition in turtle respiratory rhythm generation.

In vitro brainstem and brainstem-spinal cord preparations were used to determine the role of synaptic inhibition in respiratory rhythm generation in adult turtles. Bath application of bicuculline (a GABA(A) receptor antagonist) to brainstems increased hypoglossal burst frequency and amplitude, with peak discharge shifted towards the burst onset. Strychnine (a glycine receptor antagonist) increased amplitude and frequency, and decreased burst duration, but only at relatively high concentrations (10-100 microM). Rhythmic activity persisted during combined bicuculline and strychnine application (50 microM each) with increased amplitude and frequency, decreased burst duration, and a rapid onset-decrementing burst pattern. The bicuculline-strychnine rhythm frequency decreased during mu-opioid receptor activation or decreased bath P(C)(O(2)). Synaptic inhibition blockade in the brainstem of brainstem-spinal cord preparations increased burst amplitude in spinal expiratory (pectoralis) nerves and nearly abolished spinal inspiratory activity (serratus nerves), suggesting that medullary expiratory motoneurons were mainly active. Under conditions of synaptic inhibition blockade in vitro, the turtle respiratory network was able to produce a rhythm that was sensitive to characteristic respiratory stimuli, perhaps via an expiratory (rather than inspiratory) pacemaker-driven mechanism. Thus, these data indicate that the adult turtle respiratory rhythm generator has the potential to operate in a pacemaker-driven manner.

Group I mGluRs modulate the pattern of non-synaptic epileptiform activity in the hippocampus

The hippocampus is well known for its susceptibility to epileptic seizures, in part because of its neuronal architecture that facilitates synchronization. Although synaptic networks are important for the genesis and spread of epileptiform activity, synchronization of neuronal activity can occur when action potential-dependent chemical synaptic transmission is absent. In particular, it is possible to induce epileptiform activity by perfusing hippocampal slices with a low-Ca 2+/high-K + mediums. Using extracellular recording in area CA1 we have characterized the effects of metabotropic glutamate receptor (mGluR) activation on this non-synaptic bursting activity. Under control conditions, bursting occurred at intervals of 14–86 s with each burst comprising a long (up to 44 s) negative-going field potential of 2 to 13 mV superimposed upon which was sustained firing of population spikes. Activation of group I mGluRs by ( S)-3,5-dihydroxyphenylglycine (DHPG) (25 μM) caused a dramatic increase in burst frequency (up to five-fold), which was accompanied by a decrease in the duration and amplitude of bursts. The selective mGluR 1 antagonist 2-methyl-4-carboxyphenylglycine (LY367385) and the selective mGluR 5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) both restricted the increase in burst frequency induced by DHPG. However, only LY367385 inhibited the decrease in burst duration and amplitude. Combined application of both antagonists prevented all DHPG-induced changes in bursting activity. These data provide evidence for a role of both mGluR 1 and mGluR 5 subtypes in changing the frequency of non-synaptic bursting, with mGluR 1 alone causing alterations in burst duration and amplitude. These effects are likely to contribute to the group I mGluR-induced changes in synaptic epileptic activity that are already well documented.

Voltage-sensitive gating induced by a mutation in the fifth transmembrane domain of CFTR.

A mutation in the fifth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel (V317E) resulted in whole cell currents that exhibited marked outward rectification on expression in Xenopus oocytes. However, the single-channel unitary current (i)-voltage (V) relationship failed to account for the rectification of whole cell currents. In excised patches containing one to a few channels, the time-averaged single-channel current (I)-V relationship (I = N x P(o) x i, where N is the number of active channels and P(o) is open probability) of V317E CFTR displayed outward rectification, whereas that of wild-type CFTR was linear, indicating that the P(o) of V317E CFTR is voltage dependent. The decrease in P(o) at negative potentials was due to both a decreased burst duration and a decreased opening rate that could not be ameliorated by a 10-fold increase in ATP concentration. This behavior appears to reflect a true voltage dependence of the gating process because the P(o)-V relationship did not depend on the direction of Cl(-) movement. The results are consistent with the introduction, by a point mutation, of a novel voltage-dependent gating mode that may provide a useful tool for probing the portions of the protein that move in response to ATP-dependent gating.

PKA-mediated phosphorylation and functional modulation of the ATP-sensitive K+ channels

Adenosine 5'-triphosphate (ATP)-sensitive potassium (KATP) channels play important roles in cellular functions such as hormone secretion and excitation of muscles and neurons. KATP channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SURs). The molecular mechanism by which hormones and neurotransmitters modulate KATP channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2 subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571). Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface. Moreover, the effects of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the KATP channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.

Models of Respiratory Rhythm Generation in the Pre-Bötzinger Complex. III. Experimental Tests of Model Predictions

We used the testable predictions of mathematical models proposed by Butera et al. to evaluate cellular, synaptic, and population-level components of the hypothesis that respiratory rhythm in mammals is generated in vitro in the pre-Bötzinger complex (pre-BötC) by a heterogeneous population of pacemaker neurons coupled by fast excitatory synapses. We prepared thin brain stem slices from neonatal rats that capture the pre-BötC and maintain inspiratory-related motor activity in vitro. We recorded pacemaker neurons extracellularly and found: intrinsic bursting behavior that did not depend on Ca(2+) currents and persisted after blocking synaptic transmission; multistate behavior with transitions from quiescence to bursting and tonic spiking states as cellular excitability was increased via extracellular K(+) concentration ([K(+)](o)); a monotonic increase in burst frequency and decrease in burst duration with increasing [K(+)](o); heterogeneity among different cells sampled; and an increase in inspiratory burst duration and decrease in burst frequency by excitatory synaptic coupling in the respiratory network. These data affirm the basis for the network model, which is composed of heterogeneous pacemaker cells having a voltage-dependent burst-generating mechanism dominated by persistent Na(+) current (I(NaP)) and excitatory synaptic coupling that synchronizes cell activity. We investigated population-level activity in the pre-BötC using local "macropatch" recordings and confirmed these model predictions: pre-BötC activity preceded respiratory-related motor output by 100-400 ms, consistent with a heterogeneous pacemaker-cell population generating inspiratory rhythm in the pre-BötC; pre-BötC population burst amplitude decreased monotonically with increasing [K(+)](o) (while frequency increased), which can be attributed to pacemaker cell properties; and burst amplitude fluctuated from cycle to cycle after decreasing bilateral synaptic coupling surgically as predicted from stability analyses of the model. We conclude that the pacemaker cell and network models explain features of inspiratory rhythm generation in vitro.

Diadenosine tetraphosphate-gating of recombinant pancreatic ATP-sensitive K(+) channels.

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic beta cells where targets ATP-sensitive K(+) (K(ATP)) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic K(ATP) channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic K(ATP) channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of K(ATP) channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes K(ATP) channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.

Excitatory Role of the Hyperpolarization-Activated Inward Current in Phasic and Tonic Firing of Rat Supraoptic Neurons

The properties and functional roles of the hyperpolarization-activated inward current (I(H)) in magnocellular neurosecretory cells (MNCs) were investigated during sharp microelectrode recordings from supraoptic neurons in superfused explants of rat hypothalamus. Under current clamp, voltage responses to hyperpolarizing current pulses featured depolarizing sags that were abolished by the I(H) blocker ZD 7288. Under voltage clamp, subtraction of current responses to hyperpolarizing steps recorded in the absence and presence of ZD 7288 was used to investigate the properties of I(H). Current-voltage analysis revealed that steady-state I(H) amplitude increases with hyperpolarization, with half-maximal activation of the underlying conductance occurring at -78 mV. The time course of activation of I(H) during hyperpolarizing steps was monoexponential with time constants (100-800 msec) decreasing with hyperpolarization. The effects of ZD 7288 on I(H) were slow (tau, approximately 15 min), irreversible, and half-maximal at 1.8 micrometer. When tested on continuously active MNCs, application of 30-60 micrometer ZD 7288 caused a significant reduction in firing rate. In phasically active MNCs, the drug decreased burst duration and intraburst firing frequency and caused an increase in the duration of interburst intervals. These effects were accompanied with a small hyperpolarization of the membrane potential. In contrast, ZD 7288 had no effect on spike duration, on the amplitude of calcium-dependent afterpotentials, or on the frequencies and amplitudes of spontaneous synaptic potentials. These results confirm the presence of I(H) in MNCs of the rat supraoptic nucleus and suggest that the presence of this conductance provides an excitatory drive that contributes to phasic and tonic firing.