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1.  GABAB receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid 
The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABAB receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB.
doi:10.1098/rstb.2013.0607
PMCID: PMC4173292  PMID: 25225100
ventral tegmental area; thalamus; baclofen; reward; absence seizures
2.  Investigating local and long-range neuronal network dynamics by simultaneous optogenetics, reverse microdialysis and silicon probe recordings in vivo 
Highlights
•Optogenetics and microdialysis can be successfully combined.•How to manipulate circuits of spontaneous and evoked activities with drugs and lights?•Thalamic control of delta waves and sleep spindles.
Background
The advent of optogenetics has given neuroscientists the opportunity to excite or inhibit neuronal population activity with high temporal resolution and cellular selectivity. Thus, when combined with recordings of neuronal ensemble activity in freely moving animals optogenetics can provide an unprecedented snapshot of the contribution of neuronal assemblies to (patho)physiological conditions in vivo. Still, the combination of optogenetic and silicone probe (or tetrode) recordings does not allow investigation of the role played by voltage- and transmitter-gated channels of the opsin-transfected neurons and/or other adjacent neurons in controlling neuronal activity.
New method and results
We demonstrate that optogenetics and silicone probe recordings can be combined with intracerebral reverse microdialysis for the long-term delivery of neuroactive drugs around the optic fiber and silicone probe. In particular, we show the effect of antagonists of T-type Ca2+ channels, hyperpolarization-activated cyclic nucleotide-gated channels and metabotropic glutamate receptors on silicone probe-recorded activity of the local opsin-transfected neurons in the ventrobasal thalamus, and demonstrate the changes that the block of these thalamic channels/receptors brings about in the network dynamics of distant somatotopic cortical neuronal ensembles.
Comparison with existing methods
This is the first demonstration of successfully combining optogenetics and neuronal ensemble recordings with reverse microdialysis. This combination of techniques overcomes some of the disadvantages that are associated with the use of intracerebral injection of a drug-containing solution at the site of laser activation.
Conclusions
The combination of reverse microdialysis, silicone probe recordings and optogenetics can unravel the short and long-term effects of specific transmitter- and voltage-gated channels on laser-modulated firing at the site of optogenetic stimulation and the actions that these manipulations exert on distant neuronal populations.
doi:10.1016/j.jneumeth.2014.06.031
PMCID: PMC4164909  PMID: 25004203
T-type Ca2+ channels; HCN channels; Metabotropic glutamate receptors; EEG; Slow waves; Sleep spindles; Delta waves; Thalamus; Cortex
3.  Essential Thalamic Contribution to Slow Waves of Natural Sleep 
The Journal of Neuroscience  2013;33(50):19599-19610.
Slow waves represent one of the prominent EEG signatures of non-rapid eye movement (non-REM) sleep and are thought to play an important role in the cellular and network plasticity that occurs during this behavioral state. These slow waves of natural sleep are currently considered to be exclusively generated by intrinsic and synaptic mechanisms within neocortical territories, although a role for the thalamus in this key physiological rhythm has been suggested but never demonstrated. Combining neuronal ensemble recordings, microdialysis, and optogenetics, here we show that the block of the thalamic output to the neocortex markedly (up to 50%) decreases the frequency of slow waves recorded during non-REM sleep in freely moving, naturally sleeping-waking rats. A smaller volume of thalamic inactivation than during sleep is required for observing similar effects on EEG slow waves recorded during anesthesia, a condition in which both bursts and single action potentials of thalamocortical neurons are almost exclusively dependent on T-type calcium channels. Thalamic inactivation more strongly reduces spindles than slow waves during both anesthesia and natural sleep. Moreover, selective excitation of thalamocortical neurons strongly entrains EEG slow waves in a narrow frequency band (0.75–1.5 Hz) only when thalamic T-type calcium channels are functionally active. These results demonstrate that the thalamus finely tunes the frequency of slow waves during non-REM sleep and anesthesia, and thus provide the first conclusive evidence that a dynamic interplay of the neocortical and thalamic oscillators of slow waves is required for the full expression of this key physiological EEG rhythm.
doi:10.1523/JNEUROSCI.3169-13.2013
PMCID: PMC3858629  PMID: 24336724
4.  γ-Hydroxybutyric Acid (GHB) Is Not an Agonist of Extrasynaptic GABAA Receptors 
PLoS ONE  2013;8(11):e79062.
γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABAB receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABAA receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABAA receptors, where GHB acts as an agonist with an EC50 of 140 nM. We investigated three neuronal cell types that express a tonic GABAA receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABAA receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABAB receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.
doi:10.1371/journal.pone.0079062
PMCID: PMC3823990  PMID: 24244421
6.  Metabotropic regulation of extrasynaptic GABAA receptors 
A large body of work now shows the importance of GABAA receptor-mediated tonic inhibition in regulating CNS function. However, outside of pathological conditions, there is relatively little evidence that the magnitude of tonic inhibition is itself under regulation. Here we review the mechanisms by which tonic inhibition is known to be modulated, and outline the potential behavioral consequences of this modulation. Specifically, we address the ability of protein kinase A and C to phosphorylate the extrasynaptic receptors responsible for the tonic GABAA current, and how G-protein coupled receptors can regulate tonic inhibition through these effectors. We then speculate about the possible functional consequences of regulating the magnitude of the tonic GABAA current.
doi:10.3389/fncir.2013.00171
PMCID: PMC3829460  PMID: 24298239
extrasynaptic; GABA; kinase; tonic; plasticity
7.  Unravelling the brain targets of γ-hydroxybutyric acid 
γ-Hydroxybutyric acid (GHB) is a naturally occurring γ-aminobutyric acid (GABA) metabolite that has been proposed as a neurotransmitter/neuromodulator that acts via its own receptor (GHBR). Its exogenous administration, however, elicits central nervous system-dependent effects (e.g. memory impairment, increase in sleep stages 3 and 4, dependence, seizures and coma) that are mostly mediated by GABAB receptors. The past few years have seen important developments in our understanding of GHB neurobiology: a putative GHBR has been cloned; a transgenic model of GHB aciduria has been developed; GABAB receptor knockout mice and novel GHB analogs have helped to characterize the vast majority of exogenous GHB actions mediated by GABAB receptors; and some of the cellular mechanisms underlying the dependence/abuse properties of GHB, and its ability to elicit absence seizures and an increase in sleep stages 3 and 4, have been clarified. Nevertheless, the physiological significance of a brain GHB signaling pathway is still unknown, and there is an urgent need for a well-validated functional assay for GHBRs. Moreover, as GHB can also be metabolized to GABA, it remains to be seen whether the many GABAB receptor-mediated actions of GHB are caused by GHB itself acting directly on GABAB receptors or by a GHB-derived GABA pool (or both).
doi:10.1016/j.coph.2005.10.001
PMCID: PMC2174623  PMID: 16368267
8.  Activity of cortical and thalamic neurons during the slow (<1 Hz) rhythm in the mouse in vivo 
During NREM sleep and under certain types of anaesthesia the mammalian brain exhibits a distinctive slow (<1 Hz) rhythm. At the cellular level this rhythm correlates with so-called UP and DOWN membrane potential states. In the neocortex these UP and DOWN states correspond to periods of intense network activity and widespread neuronal silence, respectively, whereas in thalamocortical (TC) neurons, UP/DOWN states take on a more stereotypical oscillatory form with UP states commencing with a low-threshold Ca2+ potential (LTCP). Whilst these properties are now well recognized for neurons in cats and rats, whether or not they are also shared by neurons in the mouse is not fully known. To address this issue we obtained intracellular recordings from neocortical and TC neurons during the slow (<1 Hz) rhythm in anaesthetized mice. We show that UP/DOWN states in this species are broadly similar to those observed in cats and rats, with UP states in neocortical neurons being characterized by a combination of action potential output and intense synaptic activity whereas UP states in TC neurons always commence with an LTCP. In some neocortical and TC neurons we observed ‘spikelets’ during UP states, supporting the possible presence of electrical coupling. Lastly, we show that upon tonic depolarization, UP/DOWN states in TC neurons are replaced by rhythmic high-threshold (HT) bursting at ~5 Hz, as predicted by in vitro studies. Thus, UP/DOWN state generation appears to be an elemental and conserved process in mammals that underlies the slow (<1 Hz) rhythm in several species, including humans.
doi:10.1007/s00424-011-1011-9
PMCID: PMC3256325  PMID: 21892727
EEG; oscillations; sleep; neocortex; thalamocortical
10.  Self-repair in a bidirectionally coupled astrocyte-neuron (AN) system based on retrograde signaling 
In this paper we demonstrate that retrograde signaling via astrocytes may underpin self-repair in the brain. Faults manifest themselves in silent or near silent neurons caused by low transmission probability (PR) synapses; the enhancement of the transmission PR of a healthy neighboring synapse by retrograde signaling can enhance the transmission PR of the “faulty” synapse (repair). Our model of self-repair is based on recent research showing that retrograde signaling via astrocytes can increase the PR of neurotransmitter release at damaged or low transmission PR synapses. The model demonstrates that astrocytes are capable of bidirectional communication with neurons which leads to modulation of synaptic activity, and that indirect signaling through retrograde messengers such as endocannabinoids leads to modulation of synaptic transmission PR. Although our model operates at the level of cells, it provides a new research direction on brain-like self-repair which can be extended to networks of astrocytes and neurons. It also provides a biologically inspired basis for developing highly adaptive, distributed computing systems that can, at fine levels of granularity, fault detect, diagnose and self-repair autonomously, without the traditional constraint of a central fault detect/repair unit.
doi:10.3389/fncom.2012.00076
PMCID: PMC3458420  PMID: 23055965
astrocyte; calcium wave; endocannabinoid; IP3; neuron; self-repair; synapse
11.  Bidirectional Coupling between Astrocytes and Neurons Mediates Learning and Dynamic Coordination in the Brain: A Multiple Modeling Approach 
PLoS ONE  2011;6(12):e29445.
In recent years research suggests that astrocyte networks, in addition to nutrient and waste processing functions, regulate both structural and synaptic plasticity. To understand the biological mechanisms that underpin such plasticity requires the development of cell level models that capture the mutual interaction between astrocytes and neurons. This paper presents a detailed model of bidirectional signaling between astrocytes and neurons (the astrocyte-neuron model or AN model) which yields new insights into the computational role of astrocyte-neuronal coupling. From a set of modeling studies we demonstrate two significant findings. Firstly, that spatial signaling via astrocytes can relay a “learning signal” to remote synaptic sites. Results show that slow inward currents cause synchronized postsynaptic activity in remote neurons and subsequently allow Spike-Timing-Dependent Plasticity based learning to occur at the associated synapses. Secondly, that bidirectional communication between neurons and astrocytes underpins dynamic coordination between neuron clusters. Although our composite AN model is presently applied to simplified neural structures and limited to coordination between localized neurons, the principle (which embodies structural, functional and dynamic complexity), and the modeling strategy may be extended to coordination among remote neuron clusters.
doi:10.1371/journal.pone.0029445
PMCID: PMC3248449  PMID: 22242121
12.  mGluR control of interneuron output regulates feedforward tonic GABAA inhibition in the visual thalamus 
Metabotropic glutamate receptors (mGluRs) play a crucial role in regulation of phasic inhibition within the visual thalamus. Here we demonstrate that mGluR-dependent modulation of interneuron GABA release results in dynamic changes in extrasynaptic GABAA (eGABAAR) receptor-dependent tonic inhibiton in thalamocortical (TC) neurons of the rat dorsal lateral geniculate nucleus (dLGN). Application of the group I selective mGluR agonist DHPG produces a concentration-dependent enhancement of both IPSC frequency and tonic GABAA current (IGABAtonic) that is due to activation of both mGluR1a and mGluR5 subtypes. In contrast, group II/III mGluR activation decreases both IPSC frequency and IGABAtonic amplitude. Using knock-out mice we show that the mGluR-dependent modulation of IGABAtonic is dependent upon expression of δ-subunit containing eGABAARs. Furthermore, unlike the dLGN, no mGluR-dependent modulation of IGABAtonic is present in TC neurons of the somatosensory ventrobasal thalamus, which lacks GABAergic interneurons. In the dLGN, enhancement of IPSC frequency and IGABAtonic by group I mGluRs is not action potential-dependent, being insensitive to TTX, but is abolished by the L-type Ca2+ channel blocker nimodipine. These results indicate selective mGluR-dependent modulation of dendro-dendritic GABA release from F2-type terminals on interneuron dendrites and demonstrate for the first time the presence of eGABAARs on TC neuron dendritic elements that participate in ‘triadic’ circuitry within the dLGN. These findings present a plausible novel mechanism for visual contrast gain at the thalamic level and shed new light upon the potential role of glial ensheathment of synaptic triads within the dLGN.
doi:10.1523/JNEUROSCI.0317-11.2011
PMCID: PMC3130900  PMID: 21653871
dorsal lateral geniculate nucleus; interneuron; metabotropic glutamate receptor; extrasynaptic GABAA receptor; thalamocortical; glomerulus
13.  TRANSITION TO ABSENCE SEIZURES AND THE ROLE OF GABAA RECEPTORS 
Epilepsy research  2011;97(3):283-289.
Absence seizures appear to be initiated in a putative cortical ‘initiation site’ by the expression of medium-amplitude 5-9Hz oscillations, that may in part be due to a decreased phasic GABAA receptor function. These oscillations rapidly spread to other cortical areas and to the thalamus, leading to fully developed generalized spike and wave discharges. In thalamocortical neurons of genetic models, phasic GABAA inhibition is either unchanged or increased, whereas tonic GABAA inhibition is increased both in genetic and pharmacological models. This enhanced tonic inhibition is required for absence seizure generation, and in genetic models it results from a malfunction in the astrocytic GABA transporter GAT-1. Contradictory results from inbred and transgenic animals still do not allow us to draw firm conclusions on changes in phasic GABAA inhibition in the GABAergic neurons of the nucleus reticularis thalami. Mathematical modelling may enhance our understanding of these competing hypotheses, by permitting investigations of their mechanistic aspects, hence enabling a greater understanding of the processes underlying seizure generation and evolution.
doi:10.1016/j.eplepsyres.2011.07.011
PMCID: PMC3227737  PMID: 21889315
phasic GABAA inhibition; tonic GABAA inhibition; GABAB receptors; GHB; GAERS; mathematical modelling
14.  Transition to absence seizures and the role of GABAA receptors 
Epilepsy Research  2011;97(3):283-289.
Summary
Absence seizures appear to be initiated in a putative cortical ‘initiation site’ by the expression of medium-amplitude 5–9 Hz oscillations, which may in part be due to a decreased phasic GABAA receptor function. These oscillations rapidly spread to other cortical areas and to the thalamus, leading to fully developed generalized spike and wave discharges. In thalamocortical neurons of genetic models, phasic GABAA inhibition is either unchanged or increased, whereas tonic GABAA inhibition is increased both in genetic and pharmacological models. This enhanced tonic inhibition is required for absence seizure generation, and in genetic models it results from a malfunction in the astrocytic GABA transporter GAT-1. Contradictory results from inbred and transgenic animals still do not allow us to draw firm conclusions on changes in phasic GABAA inhibition in the GABAergic neurons of the nucleus reticularis thalami. Mathematical modelling may enhance our understanding of these competing hypotheses, by permitting investigations of their mechanistic aspects, hence enabling a greater understanding of the processes underlying seizure generation and evolution.
doi:10.1016/j.eplepsyres.2011.07.011
PMCID: PMC3227737  PMID: 21889315
Phasic GABAA inhibition; Tonic GABAA inhibition; GABAB receptors; GHB; GAERS; Mathematical modelling
15.  The thalamic low-threshold Ca2+ potential: a key determinant of the local and global dynamics of the slow (<1 Hz) sleep oscillation in thalamocortical networks 
During non-rapid eye movement (NREM) sleep and certain types of anaesthesia, neurons in the neocortex and thalamus exhibit a distinctive slow (<1 Hz) oscillation that consists of alternating UP and DOWN membrane potential states and which correlates with a pronounced slow (<1 Hz) rhythm in the EEG. Whilst several studies have claimed that the slow oscillation is generated exclusively in neocortical networks and then transmitted to other brain areas, substantial evidence exists to suggest that the full expression of the slow oscillation in an intact thalamocortical network requires the balanced interaction of oscillator systems in both the neocortex and thalamus. Within such a scenario, we have previously argued that the powerful low-threshold Ca2+ potential (LTCP)-mediated burst of action potentials that initiates the UP states in individual thalamocortical neurons may be a vital signal for instigating UP states in related cortical areas. To investigate these issues we constructed a computational model of the thalamocortical network which encompasses the important known aspects of the slow oscillation that have been garnered from earlier in vivo and in vitro experiments. By using this model we confirm that the overall expression of the slow oscillation is intricately reliant on intact connections between thalamus and cortex. In particular, we demonstrate that UP state-related LTCP-mediated bursts in thalamocortical neurons are proficient in triggering synchronous UP states in cortical networks, thereby bringing about a synchronous slow oscillation in the whole network. The importance of LTCP-mediated action potential bursts in the slow oscillation is also underlined by the observation that their associated dendritic Ca2+ signals are the only ones that inform corticothalamic synapses of the thalamocortical neuron output, since they, but not those elicited by tonic action potential firing, reach the distal dendritic sites where these synapses are located.
doi:10.1098/rsta.2011.0126
PMCID: PMC3173871  PMID: 21893530
thalamic neurons; cortical neurons; probabilistic network model; dendrites; intrinsic calcium signalling
16.  The thalamic low-threshold Ca2+ potential: a key determinant of the local and global dynamics of the slow (<1 Hz) sleep oscillation in thalamocortical networks 
During non-rapid eye movement sleep and certain types of anaesthesia, neurons in the neocortex and thalamus exhibit a distinctive slow (<1 Hz) oscillation that consists of alternating UP and DOWN membrane potential states and which correlates with a pronounced slow (<1 Hz) rhythm in the electroencephalogram. While several studies have claimed that the slow oscillation is generated exclusively in neocortical networks and then transmitted to other brain areas, substantial evidence exists to suggest that the full expression of the slow oscillation in an intact thalamocortical (TC) network requires the balanced interaction of oscillator systems in both the neocortex and thalamus. Within such a scenario, we have previously argued that the powerful low-threshold Ca2+ potential (LTCP)-mediated burst of action potentials that initiates the UP states in individual TC neurons may be a vital signal for instigating UP states in related cortical areas. To investigate these issues we constructed a computational model of the TC network which encompasses the important known aspects of the slow oscillation that have been garnered from earlier in vivo and in vitro experiments. Using this model we confirm that the overall expression of the slow oscillation is intricately reliant on intact connections between the thalamus and the cortex. In particular, we demonstrate that UP state-related LTCP-mediated bursts in TC neurons are proficient in triggering synchronous UP states in cortical networks, thereby bringing about a synchronous slow oscillation in the whole network. The importance of LTCP-mediated action potential bursts in the slow oscillation is also underlined by the observation that their associated dendritic Ca2+ signals are the only ones that inform corticothalamic synapses of the TC neuron output, since they, but not those elicited by tonic action potential firing, reach the distal dendritic sites where these synapses are located.
doi:10.1098/rsta.2011.0126
PMCID: PMC3173871  PMID: 21893530
thalamic neurons; cortical neurons; probabilistic network model; dendrites; intrinsic calcium signalling
17.  Infra-slow (<0.1 Hz) oscillations in thalamic relay nuclei: basic mechanisms and significance to health and disease states 
Progress in brain research  2011;193:145-162.
In the absence of external stimuli the mammalian brain continues to display a rich variety of spontaneous activity. Such activity is often highly stereotypical, invariably rhythmic and can occur with periodicities ranging from a few milliseconds to several minutes. Recently there has been a particular resurgence of interest in fluctuations in brain activity occurring at <0.1 Hz, commonly referred to as very slow or infra-slow oscillations (ISOs). Whilst this is primarily due to the emergence of functional magnetic resonance imaging (fMRI) as a technique which has revolutionised the study of human brain dynamics it is also a consequence of the application of full band electroencephalography (fbEEG). Despite these technical advances the precise mechanisms which lead to ISOs in the brain remain unclear. In a host of animal studies, one brain region that consistently shows oscillations at <0.1 Hz is the thalamus. Importantly, similar oscillations can also be observed in slices of isolated thalamic relay nuclei maintained in vitro. Here, we discuss the nature and mechanisms of these oscillations, paying particular attention to a potential role for astrocytes in their genesis. We also highlight the relationship between this activity and ongoing local network oscillations in the alpha (α) (~8-13 Hz) band, drawing clear parallels with observations made in vivo. Lastly, we consider the relevance of these thalamic ISOs to the pathological activity that occurs in certain types of epilepsy.
doi:10.1016/B978-0-444-53839-0.00010-7
PMCID: PMC3173874  PMID: 21854961
acetylcholine; metabotropic glutamate receptor; EEG; gap junctions; alpha rhythm; epilepsy; adenosine; astrocytes; GIRK channels
18.  Augmentation of Tonic GABAA Inhibition in Absence Epilepsy: Therapeutic Value of Inverse Agonists at Extrasynaptic GABAA Receptors 
It is well established that impaired GABAergic inhibition within neuronal networks can lead to hypersynchronous firing patterns that are the typical cellular hallmark of convulsive epileptic seizures. However, recent findings have highlighted that a pathological enhancement of GABAergic signalling within thalamocortical circuits is a necessary and sufficient condition for nonconvulsive typical absence seizure genesis. In particular, increased activation of extrasynaptic GABAA receptors (eGABAAR) and augmented “tonic” GABAA inhibition in thalamocortical neurons have been demonstrated across a range of genetic and pharmacological models of absence epilepsy. Moreover, evidence from monogenic mouse models (stargazer/lethargic) and the polygenic Genetic Absence Epilepsy Rats from Strasbourg (GAERS) indicate that the mechanism underlying eGABAAR gain of function is nonneuronal in nature and results from a deficiency in astrocytic GABA uptake through the GAT-1 transporter. These results challenge the existing theory that typical absence seizures are underpinned by a widespread loss of GABAergic function in thalamocortical circuits and illustrate a vital role for astrocytes in the pathology of typical absence epilepsy. Moreover, they explain why pharmacological agents that enhance GABA receptor function can initiate or exacerbate absence seizures and suggest a potential therapeutic role for inverse agonists at eGABAARs in absence epilepsy.
doi:10.1155/2011/790590
PMCID: PMC3168769  PMID: 21912539
19.  Activity of cortical and thalamic neurons during the slow (<1 Hz) rhythm in the mouse in vivo 
Pflugers Archiv  2011;463(1):73-88.
During NREM sleep and under certain types of anaesthesia, the mammalian brain exhibits a distinctive slow (<1 Hz) rhythm. At the cellular level, this rhythm correlates with so-called UP and DOWN membrane potential states. In the neocortex, these UP and DOWN states correspond to periods of intense network activity and widespread neuronal silence, respectively, whereas in thalamocortical (TC) neurons, UP/DOWN states take on a more stereotypical oscillatory form, with UP states commencing with a low-threshold Ca2+ potential (LTCP). Whilst these properties are now well recognised for neurons in cats and rats, whether or not they are also shared by neurons in the mouse is not fully known. To address this issue, we obtained intracellular recordings from neocortical and TC neurons during the slow (<1 Hz) rhythm in anaesthetised mice. We show that UP/DOWN states in this species are broadly similar to those observed in cats and rats, with UP states in neocortical neurons being characterised by a combination of action potential output and intense synaptic activity, whereas UP states in TC neurons always commence with an LTCP. In some neocortical and TC neurons, we observed ‘spikelets’ during UP states, supporting the possible presence of electrical coupling. Lastly, we show that, upon tonic depolarisation, UP/DOWN states in TC neurons are replaced by rhythmic high-threshold bursting at ~5 Hz, as predicted by in vitro studies. Thus, UP/DOWN state generation appears to be an elemental and conserved process in mammals that underlies the slow (<1 Hz) rhythm in several species, including humans.
doi:10.1007/s00424-011-1011-9
PMCID: PMC3256325  PMID: 21892727
EEG; Oscillations; Sleep; Neocortex; Thalamocortical; Electroencephalogram; T-type calcium channel; Thalamus; Neocortical neurons
20.  From sleep spindles of natural sleep to spike and wave discharges of typical absence seizures: is the hypothesis still valid? 
Pflugers Archiv  2011;463(1):201-212.
The temporal coincidence of sleep spindles and spike-and-wave discharges (SWDs) in patients with idiopathic generalized epilepsies, together with the transformation of spindles into SWDs following intramuscular injection of the weak GABAA receptor (GABAAR) antagonist, penicillin, in an experimental model, brought about the view that SWDs may represent ‘perverted’ sleep spindles. Over the last 20 years, this hypothesis has received considerable support, in particular by in vitro studies of thalamic oscillations following pharmacological/genetic manipulations of GABAARs. However, from a critical appraisal of the evidence in absence epilepsy patients and well-established models of absence epilepsy it emerges that SWDs can occur as frequently during wakefulness as during sleep, with their preferential occurrence in either one of these behavioural states often being patient dependent. Moreover, whereas the EEG expression of both SWDs and sleep spindles requires the integrity of the entire cortico-thalamo-cortical network, SWDs initiates in cortex while sleep spindles in thalamus. Furthermore, the hypothesis of a reduction in GABAAR function across the entire cortico-thalamo-cortical network as the basis for the transformation of sleep spindles into SWDs is no longer tenable. In fact, while a decreased GABAAR function may be present in some cortical layers and in the reticular thalamic nucleus, both phasic and tonic GABAAR inhibitions of thalamo-cortical neurons are either unchanged or increased in this epileptic phenotype. In summary, these differences between SWDs and sleep spindles question the view that the EEG hallmark of absence seizures results from a transformation of this EEG oscillation of natural sleep.
doi:10.1007/s00424-011-1009-3
PMCID: PMC3256322  PMID: 21861061
Epilepsy; Cortex; Thalamus; Nucleus reticularis thalami; GABA receptors
21.  Thalamic Gap Junctions Control Local Neuronal Synchrony and Influence Macroscopic Oscillation Amplitude during EEG Alpha Rhythms 
Although EEG alpha (α; 8–13 Hz) rhythms are often considered to reflect an “idling” brain state, numerous studies indicate that they are also related to many aspects of perception. Recently, we outlined a potential cellular substrate by which such aspects of perception might be linked to basic α rhythm mechanisms. This scheme relies on a specialized subset of rhythmically bursting thalamocortical (TC) neurons (high-threshold bursting cells) in the lateral geniculate nucleus (LGN) which are interconnected by gap junctions (GJs). By engaging GABAergic interneurons, that in turn inhibit conventional relay-mode TC neurons, these cells can lead to an effective temporal framing of thalamic relay-mode output. Although the role of GJs is pivotal in this scheme, evidence for their involvement in thalamic α rhythms has thus far mainly derived from experiments in in vitro slice preparations. In addition, direct anatomical evidence of neuronal GJs in the LGN is currently lacking. To address the first of these issues we tested the effects of the GJ inhibitors, carbenoxolone (CBX), and 18β-glycyrrhetinic acid (18β-GA), given directly to the LGN via reverse microdialysis, on spontaneous LGN and EEG α rhythms in behaving cats. We also examined the effect of CBX on α rhythm-related LGN unit activity. Indicative of a role for thalamic GJs in these activities, 18β-GA and CBX reversibly suppressed both LGN and EEG α rhythms, with CBX also decreasing neuronal synchrony. To address the second point, we used electron microscopy to obtain definitive ultrastructural evidence for the presence of GJs between neurons in the cat LGN. As interneurons show no phenotypic evidence of GJ coupling (i.e., dye-coupling and spikelets) we conclude that these GJs must belong to TC neurons. The potential significance of these findings for relating macroscopic changes in α rhythms to basic cellular processes is discussed.
doi:10.3389/fpsyg.2011.00193
PMCID: PMC3187667  PMID: 22007176
EEG; gap junctions; electrical synapse; alpha rhythms; acetylcholine; metabotropic glutamate receptor
22.  State-dependent firing determines intrinsic dendritic Ca2+ signalling in thalamocortical neurons 
Activity-dependent dendritic Ca2+ signals play a critical role in multiple forms of non-linear cellular output and plasticity. In thalamocortical neurons, despite the well-established spatial separation of sensory and cortical inputs onto proximal and distal dendrites respectively, little is known about the spatio-temporal dynamics of intrinsic dendritic Ca2+ signalling during the different state-dependent firing patterns that are characteristic of these neurons. Here we demonstrate that T-type Ca2+ channels are expressed throughout the entire dendritic tree of rat thalamocortical neurons and that they mediate regenerative propagation of low threshold spikes, typical of, but not exclusive to sleep states, resulting in global dendritic Ca2+ influx. In contrast, actively backpropagating action potentials, typical of wakefulness, result in smaller Ca2+ influxes that can temporally summate to produce dendritic Ca2+ accumulations which are linearly related to firing frequency but spatially confined to proximal dendritic regions. Furthermore, dendritic Ca2+ transients evoked by both action potentials and low threshold spikes are shaped by Ca2+ uptake by sarco/endoplasmic reticulum Ca2+ ATP-ases, but do not rely upon Ca2+-induced Ca2+ release. Our data demonstrate that thalamocortical neurons are endowed with intrinsic dendritic Ca2+ signalling properties that are spatially and temporally modified in a behavioural state-dependent manner, and suggest that backpropagating action potentials faithfully inform proximal sensory but not distal corticothalamic synapses of neuronal output whereas corticothalamic synapses only “detect” Ca2+ signals associated with low threshold spikes.
doi:10.1523/JNEUROSCI.2968-10.2010
PMCID: PMC3044870  PMID: 21048143
thalamocortical; dendrites; T-type calcium channel; low threshold spike; action potential; calcium extrusion
23.  Aberrant GABAA Receptor-Mediated Inhibition in Cortico-Thalamic Networks of Succinic Semialdehyde Dehydrogenase Deficient Mice 
PLoS ONE  2011;6(4):e19021.
Aberrant γ-aminobutyric acid type A (GABAA) receptor-mediated inhibition in cortico-thalamic networks remains an attractive mechanism for typical absence seizure genesis. Using the whole-cell patch clamp technique we examined ‘phasic’ and ‘tonic’ GABAA inhibition in thalamocortical neurons of somatosensory (ventrobasal, VB) thalamus, nucleus reticularis thalami (NRT) neurons, and layer 5/6 pyramidal neurons of the somatosensory (barrel) cortex of succinic semialdehyde dehydrogenase (SSADH) knock-out (SSADH−/−) mice that replicate human SSADH deficiency and exhibit typical absence seizures. We found increased sIPSC frequency in both VB and NRT neurons and larger sIPSC amplitude in VB neurons of SSADH−/− mice compared to wild-type animals, demonstrating an increase in total phasic inhibition in thalamus of SSADH−/− mice. mIPSCs in both VB and NRT neurons were no different between genotypes, although there remained a trend toward more events in SSADH−/− mice. In cortical layer 5/6 pyramidal neurons, sIPSCs were fewer but larger in SSADH−/− mice, a feature retained by mIPSCs. Tonic currents were larger in both thalamocortical neurons and layer 5/6 pyramidal neurons from SSADH−/− mice compared to WTs. These data show that enhanced, rather than compromised, GABAA receptor-mediated inhibition occurs in cortico-thalamic networks of SSADH−/− mice. In agreement with previous studies, GABAA receptor-mediated inhibitory gain-of-function may be a common feature in models of typical absence seizures, and could be of pathological importance in patients with SSADH deficiency.
doi:10.1371/journal.pone.0019021
PMCID: PMC3079762  PMID: 21526163
24.  Thalamic T-type Ca2+ channels and NREM sleep 
Cell calcium  2006;40(2):175-190.
T-type Ca2+ channels play a number of different and pivotal roles in almost every type of neuronal oscillation expressed by thalamic neurones during non-Rapid Eye Movement (NREM) sleep, including those underlying sleep theta waves, the K-complex and the slow (<1 Hz) sleep rhythm, sleep spindles and delta waves. In particular, the transient opening of T channels not only gives rise to the ‘classical’ low threshold Ca2+ potentials, and associated high frequency burst of action potentials, that are characteristically present during sleep spindles and delta waves, but also contributes to the high threshold bursts that underlie the thalamic generation of sleep theta rhythms. The persistent opening of a small fraction of T channels, i.e. ITwindow, is responsible for the large amplitude and long lasting depolarization, or UP state, of the slow (<1 Hz) sleep oscillation in thalamic neurones. These cellular findings are in part matched by the wake-sleep phenotype of global and thalamic-selective CaV3.1 knockout mice that show a decreased amount of total NREM sleep time.
T-type Ca2+ channels, therefore, constitute the single most crucial voltage-dependent conductance that permeates all activities of thalamic neurones during NREM sleep. Since ITwindow and high threshold bursts are not restricted to thalamic neurones, the cellular neurophysiology of T channels should now move away from the simplistic, though historically significant, view of these channels as being responsible only for low threshold Ca2+ potentials.
doi:10.1016/j.ceca.2006.04.022
PMCID: PMC3018590  PMID: 16777223
cortex; thalamus; slow sleep oscillations; sleep spindles; delta waves; alpha rhythm; theta rhythm; ITwindow; Ih; ICAN
25.  NeuReal: an interactive simulation system for implementing artificial dendrites and large hybrid networks 
Journal of neuroscience methods  2007;169(2):290-301.
The dynamic clamp is a technique which allows the introduction of artificial conductances into living cells. Up to now, this technique has been mainly used to add small numbers of ‘virtual’ ion channels to real cells or to construct small hybrid neuronal circuits. In this paper we describe a prototype computer system, NeuReal, that extends the dynamic clamp technique to include i) the attachment of artificial dendritic structures consisting of multiple compartments and ii) the construction of large hybrid networks comprising several hundred biophysically realistic modelled neurons. NeuReal is a fully interactive system that runs on Windows XP, is written in a combination of C++ and assembler, and uses the Microsoft DirectX application programming interface (API) to achieve high-performance graphics. By using the sampling hardware-based representation of membrane potential at all stages of computation and by employing simple look-up tables, NeuReal can simulate over 1000 independent Hodgkin and Huxley type conductances in real-time on a modern personal computer (PC). In addition, whilst not being a hard real-time system, NeuReal still offers reliable performance and tolerable jitter levels up to an update rate of 50 kHz. A key feature of NeuReal is that rather than being a simple dedicated dynamic clamp, it operates as a fast simulation system within which neurons can be specified as either real or simulated. We demonstrate the power of NeuReal with several example experiments and argue that it provides an effective tool for examining various aspects of neuronal function.
doi:10.1016/j.jneumeth.2007.10.014
PMCID: PMC3017968  PMID: 18067972
Dynamic Clamp; Thalamus; Oscillations; Computer Simulation; Gap Junctions

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