Increased Spike Frequency Research Articles

SMALL CONDUCTANCE CALCIUM‐ACTIVATED POTASSIUM CHANNELS (SK) LIMIT THE EXCITABILITY OF PVN NEURONS PROJECTING TO RVLM (PVN‐RVLM)

In vivo studies indicate that under normal condition pre‐autonomic PVN neurons are often silent or have low spontaneous activity. In spite of their importance in regulating sympathetic outflow, mechanisms that control the excitability of pre‐autonomic PVN neurons remain largely unexplored. Here, we determined the role of SK channels in limiting excitability of PVN‐RVLM neurons. In voltage‐clamp recordings, an outward current (IK) (amplitude: 72 ± 10 pA; decay constant: 234 ± 55 ms, n=8) was nearly abolished by the SK channel blocker apamin or UCL1684. Activation of IK was Ca2+ dependent and reversed at ‐97±3 mV (n=3) which was close to the EK value (‐103 mV). In current‐clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +200 pA injections and averaged 22 ± 3 Hz (n=8) and was significantly greater (P<0.05) in the presence of apamin (100 nM) (47 ± 8 Hz, n=8) or UCL1684 (100 nM) (39 ± 5, n=5). Apamin and UCL1684 each blocked a prominent medium after hyperpolarization potential (mAHP: ‐11 ± 2 mV) that was evident in response to current injection. With blockade of the mAHP, a prominent after depolarization potential (ADP) was revealed (amplitude: apamin +9 ± 1; UCL1684 +7 ± 2 mV). We conclude that SK channel activation limits the excitability of PVN‐RVLM neurons. An underlying mechanism may be the ability of the mAHP to oppose the ADP. Support: AHA0865107F (QHC) & HL076312 (GMT)

Serotonin excites fast‐spiking interneurons in the striatum

Fast-spiking interneurons (FSIs) control the output of the striatum by mediating feed-forward GABAergic inhibition of projection neurons. Their neuromodulation can therefore critically affect the operation of the basal ganglia. We studied the effects of 5-hydroxytryptamine (5-HT, serotonin), a neurotransmitter released in the striatum by fibres originating in the raphe nuclei, on FSIs recorded with whole-cell techniques in rat brain slices. Bath application of serotonin (30 μm) elicited slow, reversible depolarizations (9 ± 3 mV) in 37/46 FSIs. Similar effects were observed using conventional whole-cell and gramicidin perforated-patch techniques. The serotonin effects persisted in the presence of tetrodotoxin and were mediated by 5-HT2C receptors, as they were reversed by the 5-HT2 receptor antagonist ketanserin and by the selective 5-HT2C receptor antagonist RS 102221. Serotonin-induced depolarizations were not accompanied by a significant change in FSI input resistance. Serotonin caused the appearance of spontaneous firing in a minority (5/35) of responsive FSIs, whereas it strongly increased FSI excitability in each of the remaining responsive FSIs, significantly decreasing the latency of the first spike evoked by a current step and increasing spike frequency. Voltage-clamp experiments revealed that serotonin suppressed a current that reversed around −100 mV and displayed a marked inward rectification, a finding that explains the lack of effects of serotonin on input resistance. Consistently, the effects of serotonin were completely occluded by low concentrations of extracellular barium, which selectively blocks Kir2 channels. We concluded that the excitatory effects of serotonin on FSIs were mediated by 5-HT2C receptors and involved suppression of an inwardly rectifying K+ current.

Neuroendocrine Proopiomelanocortin Neurons Are Excited by Hypocretin/Orexin

Hypocretin/orexin, produced by a group of neurons in the lateral hypothalamus/perifornical area, enhances cognitive arousal and also may play a crucial role in modulating the neuroendocrine system. How hypocretin modulates the endocrine system remains an open question. Hypocretin cells innervate the mediobasal hypothalamus where they can potentially influence the activity of specific cell populations within the arcuate nucleus. Here, we examine whether hypocretin modulates the median eminence-projecting proopiomelanocortin (POMC) neurons identified by selective green fluorescent protein expression and antidromic stimulation or retrograde Evans blue dye tracing in transgenic mice. We find that POMC neurons, in general, and, in addition, those that project their axons to the median eminence, were robustly activated by hypocretin in a dose-dependent manner. These excitatory actions included a threefold increase in spike frequency and direct membrane depolarization of up to 22 mV (mean, 17.9+/-7.2 mV). Direct postsynaptic depolarization was decreased at more positive membrane potentials, inhibited by the sodium-calcium exchanger antagonist KB-R7943, and reduced by lowering the bath temperature, or by buffering the postsynaptic calcium with BAPTA, suggesting that the primary mechanism for hypocretin-mediated excitation is the activation of the sodium-calcium exchanger. Hypocretin also enhanced excitatory inputs to POMC cells via a presynaptic mechanism and indirectly increased the release of GABA onto these cells in a spike-dependent manner. However, these synaptic actions were not necessary to cause postsynaptic membrane depolarization and spiking. Thus, in contrast to previous suggestions that hypocretin inhibited POMC cells, our results demonstrate robust direct excitation of POMC neurons by hypocretin.

Effects of amobarbital and methohexital on epileptic activity in mesial temporal structures in epileptic patients. An EEG study with depth electrodes.

Bilateral intracarotidal Amytal (amobarbital) tests for evaluation of speech and memory function were performed during preoperative evaluation of 30 patients with drug-resistant epilepsy. In 8 of these patients (16 tests), having partial complex epilepsy, EEG was recorded with depth electrodes, implanted bilaterally in anterior mesial temporal structures. The EEGs during 13 tests could be quantified with regard to spike activity. A rapid increase in spike frequency was observed ipsilateral to the injection in all tests but one. No seizure activity or clinical seizures were provoked. This previously unnoticed effect of amobarbital could be due to a direct excitatory effect of the drug on epileptic temporal neurones or, alternatively, to a release of interictal inhibition, exerted upon these neurons by other structures. In 4 patients, the effect was compared with that of methohexital, another barbiturate known to have excitatory effects upon epileptic activity.

Into the role of insulin in neuronal activity

Event Abstract Back to Event Into the role of insulin in neuronal activity Pedro A. Lima1*, F. M. Alves1, A. Bastos1, A. M. Nascimento1, D. Ogden1 and P. F. Costa1 1 Faculdade Ciencias da Univerisidade de Lisboa, Centro de Quimica e Bioquimica, Portugal Insulin plays important functions in brain physiology and its receptors are distributed in specific brain areas such as olfactory bulb, hypothalamus and hippocampus. The role of brain insulin is unclear, although emerging data shows that insulin can enhance learning and memory. We postulate that such insulin mediated behavioural changes may result from insulin mediated alterations in neuroexcitability.We studied the effect of insulin on voltage activated potassium currents in acutely dissociated rat hippocampal CA1 pyramidal neurones and neuroblastoma cells, applying whole-cell voltage clamp techniques. Insulin inhibited slow voltage activated K+ currents (Islow) in both cell models. In CA1 neurons, the insulin sensitive current-component shows very slow inactivation (tau~1-3 s). Insulin (0.3nM) did not shift the steady state inactivation and activaction curves. Hence, insulin appears to be inhibiting Islow not via a change in channel kinetics. The channel identity of the insulin sensitive current has been determined with pharmacological tools. Most interestingly, insulin (300 nM) inhibits Islow (32.1% ±2.3; n=8) only in neurones in the post-prandrial phase (P21-P30 rats sacrificed 1 hour after eating, following overnight fasting); there was no detectable insulin evoked current reduction (up to 3 μM) in neurones from fasted rats (overnight) (n=12). The insulin effect is independent on experimental glucose availability. Preliminary data using antibodies against the InsRs suggest that the expression level is changing during the fed/fast cycle.Current–clamp recordings from CA1 pyramidal neurons conducted in fed rat brain slices showed that insulin (300 nM) determined an increase in spike frequency, and a decrease in threshold for action potential. Cell viability tests showed that insulin action in neuroexcitability is independent on the cellular processes related with insulin mediated neuroprotection. Additionally, photometric measurements and imaging from neurones loaded with Ca2+ sensitive dyes showed that insulin evoke transient increases in cytoplasmic Ca2+. This shows that insulin increases excitability in CA1 pyramidal hippocampal neurons by inhibiting a slow delayed rectifying K+ conductance, with implication to Ca2+ physiology. The relevance of such findings in the context of the insulin facilitatory effect on memory is under investigation and the significance of the insulin response dependency to the fed/fast cycle will be discussed. Conference: 11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009. Presentation Type: Poster Presentation Topic: Neuronal Communication Citation: Lima PA, Alves FM, Bastos A, Nascimento AM, Ogden D and Costa PF (2009). Into the role of insulin in neuronal activity. Front. Neurosci. Conference Abstract: 11th Meeting of the Portuguese Society for Neuroscience. doi: 10.3389/conf.neuro.01.2009.11.157 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Aug 2009; Published Online: 12 Aug 2009. * Correspondence: Pedro A Lima, Faculdade Ciencias da Univerisidade de Lisboa, Centro de Quimica e Bioquimica, Lisboa, Portugal, plima.fisio@fcm.unl.pt Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Pedro A Lima F. M Alves A. Bastos A. M Nascimento D. Ogden P. F Costa Google Pedro A Lima F. M Alves A. Bastos A. M Nascimento D. Ogden P. F Costa Google Scholar Pedro A Lima F. M Alves A. Bastos A. M Nascimento D. Ogden P. F Costa PubMed Pedro A Lima F. M Alves A. Bastos A. M Nascimento D. Ogden P. F Costa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Gastrin-releasing peptide mediates light-like resetting of the suprachiasmatic nucleus circadian pacemaker through cAMP response element-binding protein and Per1 activation.

Circadian rhythmicity in the primary mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus, is maintained by transcriptional and translational feedback loops among circadian clock genes. Photic resetting of the SCN pacemaker involves induction of the clock genes Period1 (Per1) and Period2 (Per2) and communication among distinct cell populations. Gastrin-releasing peptide (GRP) is localized to the SCN ventral retinorecipient zone, from where it may communicate photic resetting signals within the SCN network. Here, we tested the putative role of GRP as an intra-SCN light signal at the behavioral and cellular levels, and we also tested whether GRP actions are dependent on activation of the cAMP response element-binding protein (CREB) pathway and Per1. In vivo microinjections of GRP to the SCN regions of Per1::green fluorescent protein (GFP) mice during the late night induced Per1::GFP throughout the SCN, including a limited population of arginine vasopressin-immunoreactive (AVP-IR) neurons. Blocking spike-mediated communication with tetrodotoxin did not disrupt overall Per1::GFP induction but did reduce induction within AVP-IR neurons. In vitro GRP application resulted in persistent increases in the spike frequency of Per1::GFP-induced neurons. Blocking endogenous Per1 with antisense oligodeoxynucleotides inhibited GRP-induced increases in spike frequency. Furthermore, inhibition of CREB-mediated gene activation with decoy oligonucleotides blocked GRP-induced phase shifts of PER2::luciferase rhythms in SCN slices. Altogether, these results indicate that GRP communicates phase resetting signals within the SCN network via both spike-dependent and spike-independent mechanisms, and that activation of the CREB pathway and Per1 are key steps in mediating downstream events in GRP resetting of SCN neurons.

Rapid Direct Excitation and Long-Lasting Enhancement of NMDA Response by Group I Metabotropic Glutamate Receptor Activation of Hypothalamic Melanin-Concentrating Hormone Neurons

The effect of group I metabotropic glutamate receptor (mGluR1 and mGluR5) activation on identified melanin-concentrating hormone (MCH) neurons was studied using patch-clamp recording in hypothalamic slices from green fluorescent protein-expressing transgenic mice. S-3,5-dihydroxyphenylglycine (DHPG), a selective group I mGluR agonist, depolarized MCH cells and increased spike frequency. The mGluR-mediated depolarization was not blocked with tetrodotoxin but was significantly reduced by replacement of extracellular Na+ with Tris, by Ni2+ or the Na+/Ca2+ exchanger blocker KB-R7943, or with BAPTA in the pipette, consistent with a mechanism based on activation of the Na+/Ca2+ exchanger. DHPG also decreased potassium currents. DHPG-induced depolarization was reduced by either mGluR1 or mGluR5 antagonists, suggesting involvement of both receptor subtypes. DHPG-induced depolarization desensitized; blockade of mGluR1 prevented the desensitization. Group I mGluR activation enhanced NMDA-evoked currents; this enhancement was remarkably long lasting and could be blocked by protein kinase A or C blockers. DHPG potentiated electrically evoked NMDA receptor-mediated postsynaptic currents, and mGluR5 antagonists blocked this action. Group I mGluRs increased spontaneous EPSCs in MCH neurons, possibly by stimulation of nearby mGluR-expressing hypocretin neurons. We found no tonic activation of mGluRs. However, electrical stimulation produced a slow inward current, which could be blocked by group I mGluR antagonists, suggesting high, but not low, levels of synaptically released glutamate activated mGluRs. Together, group I mGluRs increase MCH neuron activity by multiple presynaptic and postsynaptic mechanisms, suggesting mGluRs may therefore play a role in hypothalamic signaling relating to MCH neuron modulation of food intake and energy metabolism.

Cannabinoids Excite Hypothalamic Melanin-Concentrating Hormone But Inhibit Hypocretin/Orexin Neurons: Implications for Cannabinoid Actions on Food Intake and Cognitive Arousal

Cannabinoids modulate energy homeostasis and decrease cognitive arousal, possibly by acting on hypothalamic neurons including those that synthesize melanin-concentrating hormone (MCH) or hypocretin/orexin. Using patch-clamp recordings, we compared the actions of cannabinoid agonists and antagonists on identified MCH or hypocretin neurons in green fluorescent protein-expressing transgenic mice. The cannabinoid type-1 receptor (CB1R) agonist R-(+)-[2,3-dihydro-5-methyl-3-(4-morpho linylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212,2) depolarized MCH cells and increased spike frequency; in contrast, WIN55,212,2 hyperpolarized and reduced spontaneous firing of the neighboring hypocretin cells, both results consistent with reduced activity seen with intracerebral cannabinoid infusions. These effects were prevented by AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a CB1R antagonist, and by tetrodotoxin, suggesting no postsynaptic effect on either neuron type. In MCH cells, depolarizing WIN55,212,2 actions were abolished by the GABA(A) receptor antagonist bicuculline, suggesting that the CB1R-mediated depolarization was attributable to reduced synaptic GABA release. WIN55,212,2 decreased spontaneous IPSCs, reduced the frequency but not amplitude of miniature IPSCs, and reduced electrically evoked synaptic currents in MCH cells. Glutamate microdrop experiments suggest that WIN55,212,2 acted on axons arising from lateral hypothalamus local inhibitory cells that innervate MCH neurons. In hypocretin neurons, the reduced spike frequency induced by WIN55,212,2 was attributable to presynaptic attenuation of glutamate release; CB1R agonists depressed spontaneous and evoked glutamatergic currents and reduced the frequency of miniature EPSCs. Cannabinoid actions on hypocretin neurons were abolished by ionotropic glutamate receptor antagonists. Together, these results show that cannabinoids have opposite effects on MCH and hypocretin neurons. These opposing actions could help explain the increase in feeding and reduction in arousal induced by cannabinoids.

Role of Adaptation in C. elegans thermotaxis. Focus on “Short-Term Adaptation and Temporal Processing in the Cryophilic Response of Caenorabditis elegans”

From honeybee foraging to bird migration, the orientation of animals in their environments is vital for survival and provides opportunities for studying the neural mechanisms that underlie the perception and processing of sensory information. Temperature is a ubiquitous environmental variable that

A bio-inspired visual collision detection mechanism for cars: Combining insect inspired neurons to create a robust system

The lobula giant movement detector (LGMD) of locusts is a visual interneuron that responds with an increasing spike frequency to an object approaching on a direct collision course. Recent studies involving the use of LGMD models to detect car collisions showed that it could detect collisions, but the neuron produced collision alerts to non-colliding, translating, stimuli in many cases. This study presents a modified model to address these problems. It shows how the neurons pre-synaptic to the LGMD show a remarkable ability to filter images, and only colliding and translating stimuli produce excitation in the neuron. It then integrates the LGMD network with models based on the elementary movement detector (EMD) neurons from the fly visual system, which are used to analyse directional excitation patterns in the biologically filtered images. Combining the information from the LGMD neuron and four directionally sensitive neurons produces a robust collision detection system for a wide range of automotive test situations.

A computational analysis of central CO2 chemosensitivity in Helix aspersa

We created a single-compartment computer model of a CO(2) chemosensory neuron using differential equations adapted from the Hodgkin-Huxley model and measurements of currents in CO(2) chemosensory neurons from Helix aspersa. We incorporated into the model two inward currents, a sodium current and a calcium current, three outward potassium currents, an A-type current (I(KA)), a delayed rectifier current (I(KDR)), a calcium-activated potassium current (I(KCa)), and a proton conductance found in invertebrate cells. All of the potassium channels were inhibited by reduced pH. We also included the pH regulatory process to mimic the effect of the sodium-hydrogen exchanger (NHE) described in these cells during hypercapnic stimulation. The model displayed chemosensory behavior (increased spike frequency during acid stimulation), and all three potassium channels participated in the chemosensory response and shaped the temporal characteristics of the response to acid stimulation. pH-dependent inhibition of I(KA) initiated the response to CO(2), but hypercapnic inhibition of I(KDR) and I(KCa) affected the duration of the excitatory response to hypercapnia. The presence or absence of NHE activity altered the chemosensory response over time and demonstrated the inadvisability of effective intracellular pH (pH(i)) regulation in cells designed to act as chemostats for acid-base regulation. The results of the model indicate that multiple channels contribute to CO(2) chemosensitivity, but the primary sensor is probably I(KA). pH(i) may be a sufficient chemosensory stimulus, but it may not be a necessary stimulus: either pH(i) or extracellular pH can be an effective stimuli if chemosensory neurons express appropriate pH-sensitive channels. The lack of pH(i) regulation is a key feature determining the neuronal activity of chemosensory cells over time, and the balanced lack of pH(i) regulation during hypercapnia probably depends on intracellular activation of pH(i) regulation but extracellular inhibition of pH(i) regulation. These general principles are applicable to all CO(2) chemosensory cells in vertebrate and invertebrate neurons.

Elevations in local gamma activity are accompanied by changes in the firing rate and information coding capacity of neurons in the region of the subthalamic nucleus in Parkinson's disease

Local field potential (LFP) gamma (55–95 Hz) activity has been recorded in the vicinity of the subthalamic nucleus with both microelectrodes and macroelectrodes in patients with Parkinson's disease undergoing functional neurosurgery. Although this activity increases with movement its functional significance remains unknown. We hypothesised that elevations in local gamma activity might be associated with an increase in the capacity of individual spike trains to code information. Changes in the median firing frequency, entropy and baud were determined during spontaneous variations in the level of simultaneously recorded LFP gamma activity in a sample of 31 neurons. The latter were recorded from the zona incerta ( n = 5) and subthalamic nucleus ( n = 26) in 10 parkinsonian patients. Although 19% of neurons showed a decrease in firing rate, overall there was a net increase in spike frequency and baud of 18.0 ± 5.5% and 16.9 ± 5.3%, when periods of high gamma were compared to periods of low gamma LFP activity. In contrast, entropy dropped by only 0.8 ± 0.2% across the sampled neuronal population during periods of high gamma. All net changes were significant. We conclude that overall there was a net elevation in firing rate and potential information coding capacity, assessed in terms of baud, amongst neurons during periods of elevated gamma LFP activity in the subthalamic region.

An Increase in AMPA and a Decrease in SK Conductance Increase Burst Firing by Different Mechanisms in a Model of a Dopamine Neuron In Vivo

A stylized, symmetric, compartmental model of a dopamine neuron in vivo shows how rate and pattern can be modulated either concurrently or differentially. If two or more parameters in the model are varied concurrently, the baseline firing rate and the extent of bursting become de-correlated, which provides an explanation for the lack of a tight correlation in vivo and is consistent with some independence of the mechanisms that generate baseline firing rates versus bursts. We hypothesize that most bursts are triggered by a barrage of synaptic input and that particularly meaningful stimuli recruit larger numbers of synapses in a more synchronous way. An example of concurrent modulation is that increasing the short-lived AMPA current evokes additional spikes without regard to pattern, producing comparable increases in spike frequency and fraction fired in bursts. On the other hand, blocking the SK current evokes additional bursts by allowing a depolarization that previously produced only a single spike to elicit two or more and elongates existing bursts by the same principle, resulting in a greater effect on pattern than rate. A probabilistic algorithm for the random insertion of spikes into the firing pattern produces a good approximation to the pattern changes induced by increasing the AMPA conductance, but not by blocking the SK current, consistent with a differential modulation in the latter case. Furthermore, blocking SK produced a longer burst with a greater intra-burst frequency in response to a simulated meaningful input, suggesting that reduction of this current may augment reward-related responses.

Nerve growth factor affects Ca2+ currents via the p75 receptor to enhance prolactin mRNA levels in GH3 rat pituitary cells

In clonal pituitary GH(3) cells, spontaneous action potentials drive the opening of Ca(v)1 (L-type) channels, leading to Ca(2+) transients that are coupled to prolactin gene transcription. Nerve growth factor (NGF) has been shown to stimulate prolactin synthesis by GH(3) cells, but the underlying mechanisms are unknown. Here we studied whether NGF influences prolactin gene expression and Ca(2+) currents. By using RT-PCR, NGF (50 ng ml(-1)) was found to augment prolactin mRNA levels by approximately 80% when applied to GH(3) cells for 3 days. A parallel change in the prolactin content was detected by Western blotting. Both NGF-induced responses were mimicked by an agonist (Bay K 8644) and prevented by a blocker (nimodipine) of L-type channels. In whole-cell patch-clamp experiments, NGF enhanced the L-type Ca(2+) current by approximately 2-fold within 60 min. This effect reversed quickly upon growth factor withdrawal, but was maintained for days in the continued presence of NGF. In addition, chronic treatment (>or= 24 h) with NGF amplified the T-type current, which flows through Ca(v)3 channels and is thought to support pacemaking activity. Thus, NGF probably increases the amount of Ca(2+) that enters per action potential and may also induce a late increase in spike frequency. MC192, a specific antibody for the p75 neurotrophin receptor, but not tyrosine kinase inhibitors (K252a and lavendustin A), blocked the effects of NGF on Ca(2+) currents. Overall, the results indicate that NGF activates the p75 receptor to cause a prolonged increase in Ca(2+) influx through L-type channels, which in turn up-regulates the prolactin mRNA.

Electrophysiological measurements in three-dimensional in vivo-mimetic organotypic cell cultures: Preliminary studies with hen embryo brain spheroids

Using three-dimensional artificial tissue constructs shown to offer organotypic functionality, hen embryo brain spheroids were used as a novel electrophysiological paradigm. For the first time, single spontaneous action potentials were recorded from spheroids in culture at day 7 in vitro (DIV) using multi-electrode arrays. At DIV14 ‘bursting behaviour’ was observed. Simple stimulation was found to induce an increase in spiking frequency with an effect that ramped up over DIV7–14. By DIV14, the frequency under stimulation was typically over twice that of the corresponding spontaneous spiking. These results indicate strong self-organizing processes in vitro within the neuronal networks of the three-dimensional spheroid cell cultures. The organotypic in vivo-mimetic nature of the spheroid paradigm was confirmed by electron microscopy that revealed an outer layer of glial cells, a glial limitans, while immunostaining for Neurofilament and Glial Fibrilliary Acidic Protein demonstrated neuronal cells with a centralized neuronal and synaptic distribution. Basic biochemical functionality was also determined and Acetylcholinesterase measured, indicating the activity of acetylcholine receptors. Thus the organotypic hen embryo brain spheroid model may offer a new paradigm in which to explore neuronal networks.

Paraoxon suppresses Ca 2+ spike and afterhyperpolarization in snail neurons: Relevance to the hyperexcitability induction

The effects of organophosphate (OP) paraoxon, active metabolite of parathion, were studied on the Ca 2+ and Ba 2+ spikes and on the excitability of the neuronal soma membranes of land snail ( Caucasotachea atrolabiata). Paraoxon (0.3 μM) reversibly decreased the duration and amplitude of Ca 2+ and Ba 2+ spikes. It also reduced the duration and the amplitude of the afterhyperpolarization (AHP) that follows spikes, leading to a significant increase in the frequency of Ca 2+ spikes. Pretreatment with atropine and hexamethonium, selective blockers of muscarinic and nicotinic receptors, respectively, did not prevent the effects of paraoxon on Ca 2+ spikes. Intracellular injection of the calcium chelator BAPTA dramatically decreased the duration and amplitude of AHP and increased the duration and frequency of Ca 2+ spikes. In the presence of BAPTA, paraoxon decreased the duration of the Ca 2+ spikes without affecting their frequency. Apamin, a neurotoxin from bee venom, known to selectively block small conductance of calcium-activated potassium channels (SK), significantly decreased the duration and amplitude of the AHP, an effect that was associated with an increase in spike frequency. In the presence of apamin, bath application of paraoxon reduced the duration of Ca 2+ spike and AHP and increased the firing frequency of nerve cells. In summary, these data suggest that exposure to submicromolar concentration of paraoxon may directly affect membrane excitability. Suppression of Ca 2+ entry during the action potential would down regulate Ca 2+-activated K + channels leading to a reduction of the AHP and an increase in cell firing.

Pharmacological and molecular enhancement of learning in aging and Alzheimer’s disease

When animals learn hippocampus-dependent associative and spatial tasks such as trace eyeblink conditioning and the water maze, CA1 hippocampal neurons become more excitable as a result of reductions in the post-burst, slow afterhyperpolarization. The calcium-activated potassium current that mediates this afterhyperpolarization is activated by the calcium influx that occurs when a series of action potentials fire and serves as a modulator of neuronal firing frequency. As a result, spike frequency accommodation is also reduced after learning. Neuronal calcium buffering processes change and/or voltage-dependent calcium currents increase during aging; leading to enhancements in the slow afterhyperpolarization, increased spike frequency accommodation and age-associated impairments in learning. We describe a series of studies done to characterize this learning-specific enhancement in intrinsic neuronal excitability and its converse in aging brain. We have also combined behavioral pharmacology and biophysics in experiments demonstrating that compounds that increase neuronal excitability in CA1 pyramidal neurons also enhance learning rate of hippocampus-dependent tasks, especially in aging animals. The studies reviewed here include those using nimodipine, an L-type calcium current blocker that tends to cross the blood-brain barrier; metrifonate, a cholinesterase inhibitor; CI1017, a muscarinic cholinergic agonist; and galantamine, a combined cholinesterase inhibitor and nicotinic agonist. Since aging is the chief risk factor for Alzheimer’s disease, a disease that targets the hippocampus and associated brain regions and markedly impairs hippocampus-dependent learning, these compounds have potential use as treatments for this disease. Galantamine has been approved by the USDA for this purpose. Finally, we have extended our studies to the TG2576 transgenic mouse model of Alzheimer’s disease (AD), that overproduces amyloid precursor protein (APP) and increases levels of toxic β-amyloid in the brain. Not only do these mice show deficits in hippocampus-dependent learning as they age, but their hippocampal neurons show a reduced capacity to increase their levels of intrinsic excitability with reductions in the slow afterhyperpolarization after application of the muscarinic agonist carbachol. These TG2576 APP overproducing mice were crossed with BACE1 knockout mice, that do not produce β-amyloid because cleavage of APP by the β-site APP cleaving enzyme 1 (BACE1) is a critical step in its formation. Not only was hippocampus-dependent learning rescued in the bigenic TG2576-BACE1 mice, but the capacity of hippocampal neurons to show normal enhancements of intrinsic excitability was restored. The series of studies reviewed here support our hypothesis that enhancement in intrinsic excitability by reductions in calcium-activated potassium currents in hippocampal neurons is an important cellular mechanism for hippocampus-dependent learning.

LTP Regulates Burst Initiation and Frequency at Mossy Fiber–Granule Cell Synapses of Rat Cerebellum: Experimental Observations and Theoretical Predictions

Long-term potentiation (LTP) is a synaptic change supposed to provide the cellular basis for learning and memory in brain neuronal circuits. Although specific LTP expression mechanisms could be critical to determine the dynamics of repetitive neurotransmission, this important issue remained largely unexplored. In this paper, we have performed whole cell patch-clamp recordings of mossy fiber-granule cell LTP in acute rat cerebellar slices and studied its computational implications with a mathematical model. During LTP, stimulation with short impulse trains at 100 Hz revealed earlier initiation of granule cell spike bursts and a smaller nonsignificant spike frequency increase. In voltage-clamp recordings, short AMPA excitatory postsynaptic current (EPSC) trains showed short-term facilitation and depression and a sustained component probably generated by spillover. During LTP, facilitation disappeared, depression accelerated, and the sustained current increased. The N-methyl-d-aspartate (NMDA) current also increased. In agreement with a presynaptic expression caused by increased release probability, similar changes were observed by raising extracellular [Ca(2+)]. A mathematical model of mossy fiber-granule cell neurotransmission showed that increasing release probability efficiently modulated the first-spike delay. Glutamate spillover, by causing tonic NMDA and AMPA receptor activation, accelerated excitatory postsynaptic potential (EPSP) temporal summation and maintained a sustained spike discharge. The effect of increasing neurotransmitter release could not be replicated by increasing receptor conductance, which, like postsynaptic manipulations enhancing intrinsic excitability, proved very effective in raising granule cell output frequency. Independent regulation of spike burst initiation and frequency during LTP may provide mechanisms for temporal recoding and gain control of afferent signals at the input stage of cerebellar cortex.

Myomodulin IncreasesIhand Inhibits the Na/K Pump to Modulate Bursting in Leech Heart Interneurons

In the medicinal leech, a rhythmically active 14-interneuron network composes the central pattern generator for heartbeat. In two segmental ganglia, bilateral pairs of reciprocally inhibitory heart interneurons (oscillator interneurons) produce a rhythm of alternating bursts of action potentials that paces activity in the pattern-generating network. The neuropeptide myomodulin decreases the period of this bursting and increases the intraburst spike frequency when applied to isolated ganglia containing these oscillator interneurons. Myomodulin also decreases period, increases spike frequency, and increases the robustness of endogenous bursting in synaptically isolated (with bicuculline) oscillator interneurons. In voltage-clamp experiments using hyperpolarizing ramps, we identify an increase in membrane conductance elicited by myomodulin with the properties of a hyperpolarization-activated current. Voltage steps confirm that myomodulin indeed increases the maximum conductance of the hyperpolarization-activated current I(h). In similar experiments using Cs(+) to block I(h), we demonstrate that myomodulin also causes a steady offset in the ramp current that is not associated with an increase in conductance. This current offset is blocked by ouabain, indicating that myomodulin inhibits the Na/K pump. In current-clamp experiments, when I(h) is blocked with Cs(+), myomodulin decreases period and increases spike frequency of alternating bursting in synaptically connected oscillator interneurons, suggesting that inhibiting the Na/K pump modulates these burst characteristics. These observations indicate that myomodulin decreases period and increases spike frequency of endogenous bursting in synaptically isolated oscillator heart interneurons and alternating bursting of reciprocally inhibitory pairs of interneurons, at least in part, by increasing I(h) and by decreasing the Na/K pump.

Direct Excitation of Hypocretin/Orexin Cells by Extracellular ATP at P2X Receptors

Hypocretin/orexin (hcrt) neurons play an important role in hypothalamic arousal and energy homeostasis. ATP may be released by neurons or glia or by pathological conditions. Here we studied the effect of extracellular ATP on hypocretin cells using whole cell patch-clamp recording in hypothalamic slices of transgenic mice expressing green fluorescent protein (GFP) exclusively in hcrt-producing cells. Local application of ATP induced a dose-dependent increase in spike frequency. In the presence of TTX, ATP (100 microM) depolarized the cells by 7.8 +/- 1.2 mV. In voltage clamp under blockade of synaptic activity with the GABA(A) receptor antagonist bicuculline, and ionotropic glutamate receptor antagonists DL-2-amino-5-phosphonopentanoic acid (AP-5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), ATP (100 microM) evoked an 18 pA inward current. The inward current was blocked by extracellular choline substitution for Na+, had a reversal potential of -27 mV, and was not affected by nominally Ca2+-free external buffer, suggesting that ATP activated a nonselective cation current. All excitatory effects of ATP showed rapid attenuation. ATP-induced excitatory actions were mimicked by nonhydrolyzable ATP-gamma-S but not by alpha,beta-MeATP and inhibited by the purinoceptor antagonists suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS). The current was potentiated by a decrease in bath pH, suggesting P2X2 subunit involvement. Frequency and amplitude of spontaneous and miniature synaptic events were not altered by ATP. Suramin, but not PPADS, caused a small suppression of evoked excitatory synaptic potentials. Together, these results show a depolarizing response to extracellular ATP that would lead to an increased activity of the hypocretin arousal system.