Synchronous Depolarization Research Articles

A Microfluidic High-Capacity Screening Platform for Neurological Disorders.

Compartmentalized cell cultures (CCCs) provide the possibility to study mechanisms of neurodegenerative diseases, such as spreading of misfolded proteins in Alzheimer's or Parkinson's disease or functional changes in, e.g., chronic pain, in vitro. However, many CCC devices do not provide the necessary capacity for identifying novel mechanisms, targets, or drugs in a drug discovery context. Here, we present a high-capacity cell culture microtiter microfluidic plate compliant with American National Standard Institute of the Society for Laboratory Automation and Screening (ANSI/SLAS) standards that allows to parallelize up to 96 CCCs/experimental units, where each experimental unit comprises three microchannel-connected compartments. The plate design allows the specific treatment of cells in individual compartments through the application of a fluidic barrier. Moreover, the compatibility of the plate with neuronal cultures was confirmed with rodent primary as well as human-induced pluripotent stem cell-derived neurons of the central or peripheral nervous system for up to 14 days in culture. Using immunocytochemistry, we demonstrated that the plate design restricts neuronal soma to individual compartments, while axons, but not dendrites, can grow through the connecting microchannels to neighboring compartments. In addition, we show that neurons are spontaneously active and, as deemed by the appearance of synchronous depolarizations in neighboring compartments, are synaptically coupled. In summary, the design of the microfluidic plate allows for both morphological and functional studies of neurological in vitro cultures with increased capacity to support identification of novel mechanisms, targets, or drugs.

PO-03-043 IMPLANTABLE CARDIOVERTER DEFIBRILLATOR THERAPY WITH LEFT BUNDLE AREA PACING DELIVERS HIGHLY SUCCESSFUL ANTITACHYCARDIA PACING THERAPY FOR VENTRICULAR TACHYCARDIA AND SUCCESSFUL DEFIBIRILLATION

Left bundle area pacing (LBAP) has emerged as a promising therapy for cardiac resynchronization. However, the effects of LBAP when used as the primary sensing and antitachycardia pacing lead for implantable cardioverter defibrillator (ICD) therapy is unknown. There are potential disadvantages of using a separate pace/sense lead as the defibrillator lead integrity alerts are not maintained and dislodgement may not be known. Additionally, DF1 leads add complexity and points of failure to an ICD system. Examine the outcomes of using a LBAP lead as the pace/sense lead in ICD therapy. A retrospective review of all patients with implanted LBAP leads for ICD therapy was performed. In all cases a DF1 defibrillator lead was used with the pace/sense portion capped and a LBAP lead used. All devices were Abbott (Ellipse, Fortify, Quadra assura). In some cases simultaneous LBAP and CS pacing was performed. Medtronic 3830 leads were used for LBAP. Outcomes included VT events, ATP, and defibrillator therapy. Programming was left ot operator discretion but when no prior history of VT was known a monitor rate of 170 and treatment rate of 200 was programmed. 65 patients were identified with 45 ICDs and 20 cardiac resynchronization therapy defibrillators (CRTDs) since January of 2020. The median follow up time was 13 months. There were 138 VT/VF episodes during the study period occuring in 10 patients. 7 episodes were adjudicated to be SVT and 2 led to inappropriate shocks (1.5%). VT cycle length was 317±31 ms (rate: 191±17 bpm) withATP was delivered in 126 episodes and successfully terminated 120 (95%) of episodes. Per patient success was 90% with an average 1.3 ATP attempts. 5 appopriate shocks were delivered with 100% success. ICD therapy with a LBAP pacing lead as the primary pace/sense lead delivers highly effective ATP and ICD therapy. The rate of successful ATP is significantly higher than previous studies of ATP which have generally ranged from 50-70%. There are several plausible reasons for this including a more rapid and synchronous depolarization of the ventricle rendering more of the ventricle refractory. The small number of shocks were successful. There were no lead dislodgements or issues with sensing. These findings merit further study into the role of LBAP for ICD and ATP therapy.

New Insights and Methods for Recording and Imaging Spontaneous Spreading Depolarizations and Seizure-Like Events in Mouse Hippocampal Slices.

Spreading depolarization (SD) is a sudden, large, and synchronous depolarization of principal cells which also involves interneurons and astrocytes. It is followed by depression of neuronal activity, and it slowly propagates across brain regions like cortex or hippocampus. SD is considered to be mechanistically relevant to migraine, epilepsy, and traumatic brain injury (TBI), but there are many questions about its basic neurophysiology and spread. Research into SD in hippocampus using slices is often used to gain insight and SD is usually triggered by a focal stimulus with or without an altered extracellular buffer. Here, we optimize an in vitro experimental model allowing us to record SD without focal stimulation, which we call spontaneous. This method uses only an altered extracellular buffer containing 0 mM Mg2+ and 5 mM K+ and makes it possible for simultaneous patch and extracellular recording in a submerged chamber plus intrinsic optical imaging in slices of either sex. We also add methods for quantification and show the quantified optical signal is much more complex than imaging alone would suggest. In brief, acute hippocampal slices were prepared with a chamber holding a submerged slice but with flow of artificial cerebrospinal fluid (aCSF) above and below, which we call interface-like. As soon as slices were placed in the chamber, aCSF with 0 Mg2+/5 K+ was used. Most mouse slices developed SD and did so in the first hour of 0 Mg2+/5 K+ aCSF exposure. In addition, prolonged bursts we call seizure-like events (SLEs) occurred, and the interactions between SD and SLEs suggest potentially important relationships. Differences between rats and mice in different chambers are described. Regarding optical imaging, SD originated in CA3 and the pattern of spread to CA1 and the dentate gyrus was similar in some ways to prior studies but also showed interesting differences. In summary, the methods are easy to use, provide new opportunities to study SD, new insights, and are inexpensive. They support previous suggestions that SD is diverse, and also suggest that participation by the dentate gyrus merits greater attention.

Human magnetism in the COVID-19 vaccination era: Rereading of a long-standing scientific fact that has resurfaced

During vaccination of the COVID-19 pandemic, distributed homemade movies —depicting metal attraction on the body— raised a question in the public mind if ingredients of vaccines play a role in observing this phenomenon. Two primary sources of magnetic fields in the human body are the heart and the brain. The reason for that is coming from a general old rule of physics that electric current creates a magnetic field. Since the heart has a more synchronous depolarization, it can produce a stronger magnetic field around 1 to 14 nT. One possible scenario, which can relate the status in the pandemic era to human magnetism, is the substantial change in the heart's electrical conduction due to infections. The scenario can be supported by frequently related myocardiopathy. The other complementary scenario may be related to the increased amount of paramagnetic substances, such as ferritin, in infected patients and macrophages of the immune system responsible for heme-iron homeostasis. Regarding the presence of biomagnetic materials in higher organisms, the most substantial challenges are the presence of a magneto-sensing protein complex called magnetoreception-related cryptochromes. More detailed investigations are needed to confirm whether the recent observed human magnetism is related to COVID-19 infection and/or vaccination or is just a coincidence. We herein propose to assess the correlation of the electrocardiogram (ECG) and echocardiogram (echo) records when strong human magnetism is observed in clinical trial studies. The relevance of the above-mentioned proteins in drug development is of great value for future pharmaceutical discussions.

Impact of gyral geometry on cortical responses to surface electrical stimulation: insights from experimental and modeling studies

Objective. Invasive simultaneous stimulation and recording from intracranial electrodes and microwire arrays were used to investigate direct cortical responses to single pulses of electrical stimulation in humans. Approach. Microwire contacts measured surface potentials in cortical microdomains at a distance of 2–6 mm from the intracranial electrode. Direct cortical responses to stimulation (<20 ms) consisted of a larger surface negative potentials. Main results. The latencies of these responses were directly or inversely correlated with distances between the intracranial electrode and microwire contacts. We hypothesize that surface negative potentials reflected local synchronous depolarization of apical dendrites of pyramidal neurons in cortical microdomains in the superficial cortical layer and resulted from the activation of gray matter axons that delivered excitatory inputs to apical dendrites after cortical stimulation. We further hypothesized that the positive or inverse distance-latency correlations of the recorded negative responses were measured depending on whether activation of neurons originated at one (crown) or multiple (crown, lip, bank) sites throughout the gyrus simultaneously. The inverse distance-latency correlations then reflected the spatiotemporal superposition of different nearby sources of neuronal recruitment in the gyrus. To prove this hypothesis, we built an anatomically informed and biophysically realistic cortical network model and simulated early responses of cortical neurons to electrical stimulation in this cortical network model. The model simulations yielded negative potentials in simulated microdomains in the cortical model consistent with those recorded from humans. The model predicted sensitivity of cortical responses to the alignment of the stimulating electrode and microwire array with respect to the cortical gyrus and confirmed that gyral geometry has a major impact on direct neuronal recruitment, the timing, and the time course of neuronal activation in cortical microdomains. Significance. In this work, we demonstrated how the high-resolution forward network models can be used for better understanding and detailed prediction of cortical stimulation effects. Accurate predictive modeling tools are needed for the progress of brain stimulation therapies.

S6. SLEEP ENDOPHENOTYPES OF SCHIZOPHRENIA: A HIGH-DENSITY EEG STUDY IN DRUG-NAïVE, FIRST EPISODE PSYCHOSIS PATIENTS

BackgroundSlow waves, the hallmark of the deep nonrapid eye movement sleep electroencephalogram (EEG), are critical for restorative sleep and brain plasticity. They arise from the synchronous depolarization and hyperpolarization of millions of cortical neurons and their proper generation and propagation relies upon the integrity of widespread cortico-thalamic networks. Slow wave abnormalities have been reported in patient with Schizophrenia, although with partially contradictory results, probably related to antipsychotic and sedative medications. Recently, their presence and delineation, have been convincingly shown in first-episode psychosis patients (FEP). However, clear evidence of this biomarker at the onset of the disease, prior to any psychopharmacological intervention, remains limited. Moreover, no attempt has been made to elucidate the prognostic meaning of this finding.MethodsWe collected whole night sleep high–density electroencephalography recordings (64-channel BrainAmp, Brain Products GmbH, Gilching, Germany) in 20 drug-naive FEP patients and 20 healthy control subjects (HC). Several clinical psychometric scales as well as neurocognitive tests were administered to all subjects in order to better define psychopathological status and vulnerability. EEG slow wave activity (SWA, spectral power between 1 and 4 Hz) and several slow wave parameters were computed at each electrode location, including density and amplitude, at each electrode location. Along with a group analysis between FEP and HC, a subgroup analysis was also computed between patients who showed a progression of symptoms to full-blown Schizophrenia (SCZ, n = 10) over the next 12-month follow-up and those who did not (OTH, n = 10).ResultsSleep macro-architecture was globally preserved in FEP patients. SWA (1–4 Hz) was lower in FEP compared to HC but this difference didn’t reach statistical significance. Slow wave density was decreased in FEP compared to HC, with a significance that survived multiple comparison correction over a large fronto-central cluster. Mean amplitude was preserved. At the subgroup analysis, these results were largely driven by the subgroup of patients with a confirmed diagnosis of SCZ at a 12-month follow-up. Indeed, no difference could be found between OTH and HC, while a strong significance was still evident between SCZ and HC.DiscussionOur data confirm previous findings on reduced slow wave density in FEP, and expand them to acute subjects, before any treatment is prescribed. This is in line with available data on diffuse abnormalities of cortico-cortical and cortico-thalamic networks in these patients. Interestingly, our data also offer preliminary evidence that this deficit is specific for SCZ, as it appears to differentiate patients who developed SCZ from those with other diagnoses at follow-up. Given the traveling properties of slow waves, future research should establish their potential as markers of connectivity in SCZ.

Intraventricular Delay and Blocks.

From the atrioventricular node, electrical activation is propagated to both ventricles by a system of specialized conducting fibers, His Purkinje System (HPS), guaranteeing a fast, synchronous depolarization of both ventricles. From the predivisional common stem, a right and left branch separate, subdividing further in a fairly predictable fashion. Synchronous ventricular activation results in a QRS with specific characteristics and duration of less than 110 milliseconds. Block or delay in any part of the HPS changes the electrocardiographic (ECG) morphology. This article discusses the use and limitations of standard ECG in detecting abnormal ventricular propagation in specific areas of the HPS.

Postsynaptic GABA(B) Receptors Contribute to the Termination of Giant Depolarizing Potentials in CA3 Neonatal Rat Hippocampus.

During development, hippocampal CA3 network generates recurrent population bursts, so-called Giant Depolarizing Potentials (GDPs). GDPs are characterized by synchronous depolarization and firing of CA3 pyramidal cells followed by afterhyperpolarization (GDP-AHP). Here, we explored the properties of GDP-AHP in CA3 pyramidal cells using gramicidin perforated patch clamp recordings from neonatal rat hippocampal slices. We found that GDP-AHP occurs independently of whether CA3 pyramidal cells fire action potentials (APs) or remain silent during GDPs. However, the amplitude of GDP-AHP increased with the number of APs the cells fired during GDPs. The reversal potential of the GDP-AHP was close to the potassium equilibrium potential. During voltage-clamp recordings, current-voltage relationships of the postsynaptic currents activated during GDP-AHP were characterized by reversal near the potassium equilibrium potential and inward rectification, similar to the responses evoked by the GABA(B) receptor agonists. Finally, the GABA(B) receptor antagonist CGP55845 strongly reduced GDP-AHP and prolonged GDPs, eventually transforming them to the interictal and ictal-like discharges. Together, our findings suggest that the GDP-AHP involves two mechanisms: (i) postsynaptic GABA(B) receptor activated potassium currents, which are activated independently on whether the cell fires or not during GDPs; and (ii) activity-dependent, likely calcium activated potassium currents, whose contribution to the GDP-AHP is dependent on the amount of firing during GDPs. We propose that these two complementary inhibitory postsynaptic mechanisms cooperate in the termination of GDP.

Asynchronous presynaptic glutamate release enhances neuronal excitability during the post-spike refractory period.

Many excitatory synapses in the brain release glutamate with both synchronous and asynchronous components. Immediately following an action potential, neurons display a reduced excitability due to the post-spike afterhyperpolarization (AHP). This gives rise to a relative refractory period. When an action potential is evoked by glutamate synaptic input possessing asynchronous release, the delayed glutamate release events act to depolarize the neuron during the AHP and overcome the relative refractory period. These results demonstrate a new role for asynchronous release in regulating post-spike excitability and the relative refractory period in central neurons. Post-spike afterhyperpolarizations (AHPs) functionally inhibit neuronal excitability for tens to hundreds of milliseconds following each action potential. This imposes a relative refractory period during which synaptic excitation is less effective at evoking spikes. Here we asked whether some synapses have mechanisms in place that allow them to overcome the AHP and drive spiking in target cells during this period of reduced excitability. We examined glutamate synapses onto oxytocin and vasopressin neurons in the paraventricular nucleus of the hypothalamus. These synapses can display pronounced asynchronous glutamate release following a single presynaptic spike, with the time course of release being similar to that of the post-spike AHP. To test whether asynchronous release is more effective at overcoming the relative refractory period, we evoked a single action potential with either a brief synchronous depolarization or an asynchronous potential and then assessed excitability at multiple time points following the spike. Neurons receiving asynchronous depolarizing synaptic inputs had a shorter relative refractory period than those receiving synchronous depolarizations. Our data demonstrate that synapses releasing glutamate in an asynchronous and delayed manner are ideally adapted to counter the AHP. By effectively overcoming the relative refractory period, the kinetics of excitatory synaptic input can play an important role in controlling post-spike excitability.

Multifaceted roles for astrocytes in spreading depolarization: A target for limiting spreading depolarization in acute brain injury?

Spreading depolarizations (SDs) are coordinated waves of synchronous depolarization, involving large numbers of neurons and astrocytes as they spread slowly through brain tissue. The recent identification of SDs as likely contributors to pathophysiology in human subjects has led to a significant increase in interest in SD mechanisms, and possible approaches to limit the numbers of SDs or their deleterious consequences in injured brain. Astrocytes regulate many events associated with SD. SD initiation and propagation is dependent on extracellular accumulation of K(+) and glutamate, both of which involve astrocytic clearance. SDs are extremely metabolically demanding events, and signaling through astrocyte networks is likely central to the dramatic increase in regional blood flow that accompanies SD in otherwise healthy tissues. Astrocytes may provide metabolic support to neurons following SD, and may provide a source of adenosine that inhibits neuronal activity following SD. It is also possible that astrocytes contribute to the pathophysiology of SD, as a consequence of excessive glutamate release, facilitation of NMDA receptor activation, brain edema due to astrocyte swelling, or disrupted coupling to appropriate vascular responses after SD. Direct or indirect evidence has accumulated implicating astrocytes in many of these responses, but much remains unknown about their specific contributions, especially in the context of injury. Conversion of astrocytes to a reactive phenotype is a prominent feature of injured brain, and recent work suggests that the different functional properties of reactive astrocytes could be targeted to limit SDs in pathophysiological conditions.

Ontogeny of rhythmic breathing‐related motor activity in the Zebra Finch brainstem

Activity in the CNS begins as a widely distributed and synchronous depolarization wave known as rhythmic spontaneous neural activity (rSNA). As growth and maturation proceed, rSNA differentiates into central pattern generators (CPGs) that produce individual rhythmic behaviors, such as breathing. While it is important to understand the mechanisms that underlie this progression, monitoring the uninterrupted development of breathing‐related motor circuits remains difficult due to the limited access of these circuits in the mammalian model and a lack of information about breathing‐related CPG behavior in other models, such as birds. Here, we use a novel brainstem spinal cord preparation from the altricial Zebra Finch embryo to study the early development and maturation of breathing‐related motor patterns conveyed in cranial nerve IX. We show that rSNA can be measured continuously from embryonic day 4 (E4) through external pipping (E14). rSNA starts at embryonic day 4 (E4) with low frequency (0.29 bursts/min) short duration (&lt;2.5 sec) single bursts, and progresses to a more complex pattern consisting of episodes with short duration bursts coupled to slow frequency (0.2 burst/minute) long duration bursts (&gt;80 sec) at E9. The activity returns to short duration (&lt;2.5 sec), high frequency (6‐12 bursts/min) at hatching (E14). At E4 the activity is nicotinergic‐sensitive and in the older animals (&gt;E7) the short duration bursts are completely blocked by NMDA and non‐NMDA antagonism. The long duration bursts only diminished by bath application of m‐type glutamate antagonists. These findings establish an extremely tractable new experimental model to study the development of central breathing behaviors in both health and disease.

Substitution of extracellular Ca2+ by Sr2+ prolongs inspiratory burst in pre-Bötzinger complex inspiratory neurons.

The pre-Bötzinger complex (preBötC) underlies inspiratory rhythm generation. As a result of network interactions, preBötC neurons burst synchronously to produce rhythmic premotor inspiratory activity. Each inspiratory burst consists of action potentials (APs) on top of a 10- to 20-mV synchronous depolarization lasting 0.3-0.8 s known as inspiratory drive potential. The mechanisms underlying the initiation and termination of the inspiratory burst are unclear, and the role of Ca(2+) is a matter of intense debate. To investigate the role of extracellular Ca(2+) in inspiratory burst initiation and termination, we substituted extracellular Ca(2+) with Sr(2+). We found for the first time an ionic manipulation that significantly interferes with burst termination. In a rhythmically active slice, we current-clamped preBötC neurons (Vm ≅ -60 mV) while recording integrated hypoglossal nerve (∫XIIn) activity as motor output. Substitution of extracellular Ca(2+) with either 1.5 or 2.5 mM Sr(2+) significantly prolonged the duration of inspiratory bursts from 653.4 ± 30.7 ms in control conditions to 981.6 ± 78.5 ms in 1.5 mM Sr(2+) and 2,048.2 ± 448.5 ms in 2.5 mM Sr(2+), with a concomitant increase in decay time and area. Substitution of extracellular Ca(2+) by Sr(2+) is a well-established method to desynchronize neurotransmitter release. Our findings suggest that the increase in inspiratory burst duration is determined by a presynaptic mechanism involving desynchronization of glutamate release within the network.

Getting at the heart of the issue: advanced imaging and stem cell‐based therapy offer a novel approach to cardiac resynchronization

The pump function of the left ventricle (LV) is dependent on synchronous depolarization and contraction to expel blood efficiently. The propagation of the apical-to-basal ventricular contraction pattern is controlled by electrical signalling of the fibre system down the interventricular septum and woven through the free walls of the ventricular myocardium. Dyssynchronous contraction results in mechanical inefficiency, decreased external work, and reduced cardiac output. Myocardial infarction (MI) may cause wall motion abnormalities not only through disturbed electrical conduction, but also through myocardial damage and non-viable tissue. Therapy targeted at preventing adverse remodelling and improving the viability of non-contracting tissue may have the potential for great improvement in ventricular synchronization by addressing mechanical disturbances in the myocardium. Recent advances in stem cell therapy hold promise for regenerative cardiac interventions, as the use of undifferentiated stem cells delivered to the myocardium may be effective in forming functional cardiac tissue and restoring global heart function. While the potential use of such therapies has gained recent attention, the administration of stem cell therapy to ameliorate regional dyssynchrony related to MI has not been examined.

OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange

The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ∼0.4 pH units that persist in cells permeabilized in ion-free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro-fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro-fission Dynamin-related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria.

Dopamine suppresses persistent network activity via D1‐like dopamine receptors in rat medial entorhinal cortex

Cortical networks display persistent activity in the form of periods of sustained synchronous depolarizations (‘UP states’) punctuated by periods of relative hyperpolarization (‘DOWN states’), which together form the slow oscillation. UP states are known to be synaptically generated and are sustained by a dynamic balance of excitation and inhibition, with fast ionotropic glutamatergic excitatory and GABAergic inhibitory conductances increasing during the UP state. Previously, work from our group demonstrated that slow metabotropic GABA receptors also play an important role in terminating the UP state, but the effects of other neuromodulators on this network phenomenon have received little attention. Given that persistent activity is a neural correlate of working memory and that signalling through dopamine receptors has been shown to be critical for working memory tasks, we examined whether dopaminergic neurotransmission affected the slow oscillation. Here, using an in vitro model of the slow oscillation in rat medial entorhinal cortex, we showed that dopamine strongly and reversibly suppressed cortical UP states. We showed that this effect was mediated through D1-like and not D2-like dopamine receptors, and we found no evidence that tonic dopaminergic transmission affected UP states in our model.

Neonatal Hypoxia and Seizures

1. Maria Gillam-Krakauer, MD* 2. Brian S. Carter, MD† 1. *Assistant Professor of Pediatrics, Division of Neonatology, Vanderbilt University Medical Center, Nashville, TN. 2. †Professor of Pediatrics, Section of Neonatology, Children's Mercy Hospital, Kansas City, MO. * Abbreviations: AED: : antiepileptic drug AMPA: : α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid ATP: : adenosine triphosphate CSF: : cerebrospinal fluid HIE: : hypoxic-ischemic encephalopathy MOCO: : molybdenum cofactor NMDA: : N -methyl-d-aspartic acid With 1 to 3 in 1,000 term neonates experiencing seizures, pediatricians need to know how to determine the seizure cause and manage appropriately, using brain imaging and treatments such as therapeutic hypothermia, xenon, and other pharmacologic therapies, in order to minimize long-term sequelae and leverage the infant brain's tremendous capacity for repair in the first 2 years after birth. After completing this article, readers should be able to: 1. Understand the pathophysiology of neonatal seizures. 2. Know the many disorders associated with seizures in the newborn. 3. Be aware of the characteristics of different neonatal seizure syndromes. 4. Know how to evaluate a newborn who is having seizures. 5. Be aware of the treatments for neonatal seizures. 6. Understand the characteristics and management of hypoxic-ischemic encephalopathy. Seizures occur during the newborn period at an incidence of ∼1 to 3 per 1,000 infants born at term. (1)(2)(3) Numerous systemic and neurologic conditions can manifest as seizures. Cerebral hypoxia-ischemia, defined as partial lack of oxygen resulting in reduction of blood flow to the brain, is the most frequent cause of seizures in the newborn period. It is important to determine the cause of neonatal seizures and institute the appropriate therapy to minimize the long-term sequelae of both the underlying condition and the seizure. Seizures are paroxysmal alterations in neurologic function caused by excessive synchronous depolarization of neurons within the central nervous system. Regardless of the underlying pathology manifesting as a seizure, all seizures are due to a shift in cell energy. This shift can result from failure of the adenosine triphosphate (ATP)–dependent sodium-potassium (Na+-K+) pump, an imbalance of inhibitory and excitatory neurotransmitters, and both excessive …

Inhibition of endoplasmic reticulum Ca2+ ATPase in preBötzinger complex of neonatal rat does not affect respiratory rhythm generation

PreBötzinger complex (preBötC) neurons in the brainstem underlie respiratory rhythm generation in vitro. As a result of network interactions, preBötC neurons burst synchronously to produce rhythmic premotor inspiratory activity. Each inspiratory neuron has a characteristic 10–20mV, 0.3–0.8 s synchronous depolarization known as the inspiratory drive potential or inspiratory envelope, topped by action potentials (APs). Mechanisms involving Ca2+ fluxes have been proposed to underlie the initiation of the inspiratory drive potential. An important source of intracellular Ca2+ is the endoplasmic reticulum (ER) in which active Ca2+ sequestration is mediated by a class of transporters termed sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs). We aim to test the hypothesis that disruption of Ca2+ sequestration into the ER affects respiratory rhythm generation. We examined the effect of inhibiting SERCA on respiratory rhythm generation in an in vitro slice preparation. Bath application of the potent SERCA inhibitors thapsigargin or cyclopiazonic acid (CPA) for up to 90min did not significantly affect the period or amplitude of respiratory-related motor output or integral and duration of inspiratory drive in preBötC neurons. We promoted the depletion of intracellular Ca2+ stores by a transient bath application of 30mM K+ (high K+) in the continuous presence of thapsigargin or CPA. After washing out the high K+, respiratory rhythm period and amplitude returned to baseline values. These results show that after inhibition of SERCA and depletion of intracellular Ca2+ stores, respiratory rhythm remains substantially the same, suggesting that this source of Ca2+ does not significantly contribute to rhythm generation in the preBötC in vitro.

Dysregulation of Cardiogenesis, Cardiac Conduction, and Cell Cycle in Mice Lacking miRNA-1-2

MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the expression of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in mammals through targeted deletion remains unknown, and reliable prediction of mRNA targets is still problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell-cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 "seed matches" and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets.

Fictive locomotor patterns generated by tetraethylammonium application to the neonatal rat spinal cord in vitro

Intrinsic spinal networks generate a locomotor rhythm characterized by alternating electrical discharges from flexor and extensor motor pools. Because this process is preserved in the rat isolated spinal cord, this preparation in vitro may be a useful model to explore methods to reactivate locomotor networks damaged by spinal injury. The present electrophysiological investigation examined whether the broad spectrum potassium channel blocker tetraethylammonium could generate locomotor-like patterns. Low (50–500μM) concentrations of tetraethylammonium induced irregular, synchronous discharges incompatible with locomotion. Higher concentrations (1–10mM) evoked alternating discharges between flexor and extensor motor pools, plus large depolarization of motoneurons with spike broadening. The alternating discharges were superimposed on slow, shallow waves of synchronous depolarization. Rhythmic alternating patterns were suppressed by blockers of glutamate, GABA A and glycine receptors, disclosing a background of depolarizing bursts inhibited by antagonism of group I metabotropic glutamate receptors. Furthermore, tetraethylammonium also evoked irregular discharges on dorsal roots. Rhythmic alternating patterns elicited by tetraethylammonium on ventral roots were relatively stereotypic, had limited synergy with fictive locomotion induced by dorsal root stimuli, and were not accelerated by 4-aminopyridine. Horizontal section of the spinal cord preserved irregular ventral root discharges and dorsal root discharges, demonstrating that the action of tetraethylammonium on spinal networks was fundamentally different from that of 4-aminopyridine. These results show that a potassium channel blocker such as tetraethylammonium could activate fictive locomotion in the rat isolated spinal cord, although the pattern quality lacked certain features like frequency modulation and strong synergy with other inputs to locomotor networks.

Organization of Interlaminar Interactions in the Rat Superior Colliculus

Our previous studies have shown that when slices of the rat superior colliculus (SC) are exposed to a solution containing 10 microM bicuculline and a low concentration of Mg2+ (0.1 mM), most neurons in the intermediate gray layer (stratum griseum intermediale; SGI), wide-field vertical (WFV) cells in the optic layer (stratum opticum; SO), and a minor population of neurons in the superficial gray layer (stratum griseum superficiale; SGS) exhibit spontaneous depolarization and burst firing, which are synchronous among adjacent neurons. These spontaneous and synchronous depolarizations were thought to share common mechanisms with presaccadic burst activity in SGI neurons. In the present study, we explored the site responsible for generation of synchronous depolarization of SGI neurons by performing dual whole cell recordings under different slice conditions. A pair of SGI neurons recorded in a small rectangular piece of the SGI punched out from the SC slice showed synchronous depolarization but far less frequently than those recorded in a small rectangular piece including SGS and SO. This suggests that the superficial layers are needed for triggering synchronous depolarization in the SGI. Furthermore, we recorded spontaneous depolarizations in pairs of neurons belonging to the different layers. Analysis of their synchronicity revealed that WFV cells in the SO exhibit synchronous depolarizations with both SGS and SGI neurons, and the onset of spontaneous depolarization in WFV cells precedes those of neurons in other layers. Further, when SGS and SGI neurons exhibit synchronous depolarizations, SGI neurons usually precede the SGS neurons. These observations give further evidence to the existence of interlaminar interaction between superficial and deeper layers of the SC. In addition, it is suggested that WFV cells can trigger burst activity in other layers of the SC and that there is an excitatory signal transmission from the deeper layers to the superficial layers.