Dynamics of sleep oscillations is coupled to brain temperature on multiple scales.

KEY POINTS: Sleep spindle frequency positively, duration negatively correlates with brain temperature. Local heating of the thalamus produces similar effects in the heated area. Thalamic network model corroborates temperature dependence of sleep spindle frequency. Brain temperature shows spontaneous microfluctuations during both anesthesia and natural sleep. Larger fluctuations are associated with epochs of REM sleep. Smaller fluctuations correspond to the alteration of spindling and delta epochs of infra-slow oscillation. ABSTRACT: Every form of neural activity depends on temperature, yet its relationship to brain rhythms is poorly understood. In this work we examined how sleep spindles are influenced by changing brain temperatures and how brain temperature is influenced by sleep oscillations. We employed a novel thermoelectrode designed for measuring temperature while recording neural activity. We found that spindle frequency is positively correlated and duration negatively correlated with brain temperature. Local heating of the thalamus replicated the temperature dependence of spindle parameters in the heated area only, suggesting biophysical rather than global modulatory mechanisms, a finding also supported by a thalamic network model. Finally, we show that switches between oscillatory states also influence brain temperature on a shorter and smaller scale. Epochs of paradoxical sleep as well as the infra-slow oscillation were associated with brain temperature fluctuations below 0.2°C. Our results highlight that brain temperature is massively intertwined with sleep oscillations on various time scales.

Intrathalamic connections shape spindle activity - a modelling study.

Spindle oscillations are generated predominantly during sleep state II, through cyclical interactions between thalamocortical and reticular neurons. Inhibition from reticular cells is critical for this activity; it enables burst firing by the de-inactivation of T-type Ca2+ channels. While the effect of different channelopathies on spindling is extensively investigated, our knowledge about the role of intrathalamic connections is limited. Therefore, we explored how the connection pattern and the density of reticular inhibitory synapses affect spindle activity in a thalamic network model. With more intrareticular connections, synchronous firing of reticular cells, and intraspindle burst frequency decreased, spindles lengthened. In models with strong intrareticular inhibition spindle activity was impaired, and a sustained 6-8 Hz oscillation was generated instead. The strength of reticular innervation onto thalamocortical cells played a key role in the generation of oscillations; it determined the amount of thalamocortical cell bursts, and consequently spindle length. Focal inputs supported bursts but affected only a few cells thus barely reinforced network activity, while diffuse contacts aided bursts only when a sufficient number of reticular cells fired synchronously. According to our study, alterations in the connection pattern influence thalamic activities and may contribute to pathological conditions, or alternatively, they serve as a compensatory mechanism.

GABA(B) 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 GABA(B) receptors (GABA(B)Rs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABA(A) and GABA(B)Rs, 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 GABA(B)R agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca(2+) in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABA(B)Rs and mediated by Ca(2+) 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.

Activation of astroglial calcium signaling by endogenous metabolites succinate and gamma-hydroxybutyrate in the nucleus accumbens.

Accumulating evidence suggests that different energy metabolites play a role not only in neuronal but also in glial signaling. Recently, astroglial Ca(2+) transients evoked by the major citric acid cycle metabolite succinate (SUC) and gamma-hydroxybutyrate (GHB) that enters the citric acid cycle via SUC have been described in the brain reward area, the nucleus accumbens (NAc). Cells responding to SUC by Ca(2+) transient constitute a subset of ATP-responsive astrocytes that are activated in a neuron-independent way. In this study we show that GHB-evoked Ca(2+) transients were also found to constitute a subset of ATP-responsive astrocytes in the NAc. Repetitive Ca(2+) dynamics evoked by GHB suggested that Ca(2+) was released from internal stores. Similarly to SUC, the GHB response was also characterized by an effective concentration of 50 µM. We observed that the number of ATP-responsive cells decreased with increasing concentration of either SUC or GHB. Moreover, the concentration dependence of the number of ATP-responsive cells were highly identical as a function of both [SUC] and [GHB], suggesting a mutual receptor for SUC and GHB, therefore implying the existence of a distinct GHB-recognizing astroglial SUC receptor in the brain. The SUC-evoked Ca(2+) signal remained in mice lacking GABA(B) receptor type 1 subunit in the presence and absence of the N-Methyl-d-Aspartate (NMDA) receptor antagonist (2R)-amino-5-phosphonovaleric acid (APV), indicating action mechanisms independent of the GABA(B) or NMDA receptor subtypes. By molecular docking calculations we found that residues R99, H103, R252, and R281 of the binding crevice of the kidney SUC-responsive membrane receptor SUCNR1 (GPCR91) also predict interaction with GHB, further implying similar GHB and SUC action mechanisms. We conclude that the astroglial action of SUC and GHB may represent a link between brain energy states and Ca(2+) signaling in astrocytic networks.

Calcium signals in the nucleus accumbens: activation of astrocytes by ATP and succinate.

BACKGROUND: Accumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc), and thereby to explore the action of citric acid cycle intermediate succinate (SUC). RESULTS: We described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43) and the glial fibrillary acidic protein (GFAP) indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthio)adenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 µM) in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously. CONCLUSIONS: We concluded, therefore, that ATP- and SUC-sensitive Ca2+ transients appear to represent a signalling layer independent of NAc neurons. This previously unrecognised glial action of SUC, a major cellular energy metabolite, may play a role in linking metabolism to Ca2+ signalling in astrocytic networks under physiological and pathological conditions such as exercise and metabolic diseases.

GABAergic signaling in primary lens epithelial and lentoid cells and its involvement in intracellular Ca2+ modulation.

Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouse lenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on the intracellular Ca2+ ([Ca2+]i) level. We have found that these cultures express specific cellular markers for lens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thus recapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-B receptors (GABAAR and GABABR) with the specific agonists muscimol and baclofen, respectively induces [Ca2+]i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and were dependent on extracellular Ca2+. Bicuculline did not change the GABA-evoked Ca2+ responses in Ca2-containing buffers, but suppressed them significantly in Ca2+-free buffers suggesting the two receptors couple to convergent Ca2+ mobilization mechanisms with different extracellular Ca2+ sensitivity. Prolonged activation of GABABR induced wave propagation of the Ca2+ signal and persistent oscillations. The number of cells reacting to GABA or GABA+bicuculline in P30 mouse LEC cultures expressing predominantly the synaptic type GABAAR did not differ significantly from the number of reacting cells in P0 mouse LEC cultures. The GABA-induced Ca2+ transients in P30 (but not P0) mouse LEC could be entirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca2+ signaling may be involved in a variety of Ca2+-dependent cellular processes during lens growth and epithelial cell differentiation.

GABA(A) and GABA(B) receptors of distinct properties affect oppositely the proliferation of mouse embryonic stem cells through synergistic elevation of intracellular Ca(2+).

Gamma-amminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system of vertebrates, serves as an autocrine/paracrine signaling molecule during development, modulating a number of calcium (Ca(2+))-dependent processes, including proliferation, migration, and differentiation, acting via 2 types of GABA receptors (GABARs): ionotropic GABA(A)Rs and metabotropic GABA(B)Rs. Here, we demonstrate that mouse embryonic stem cells (mESCs), which possess the capacity for virtually unlimited self-renewal and pluripotency, synthesize GABA and express functional GABA(A)Rs and GABA(B)Rs, as well as voltage-gated calcium channels (VGCCs), ryanodine receptors (RyRs), and inwardly rectifying potassium (GIRK) channels. On activation, both GABAR types triggered synergistically intracellular calcium rise. Muscimol (a GABA(A)R agonist) induced single Ca(2+) transients involving both VGCC-mediated Ca(2+) influx and intracellular stores, while baclofen (a GABA(B)R agonist) evoked Ca(2+) transients followed by intercellular Ca(2+) waves and oscillations that were resistant to antagonists and entirely dependent on Ca(2+) release from intracellular stores. Prolonged treatment with muscimol slightly inhibited, while baclofen or SR95531 (a GABA(A)R antagonist) significantly facilitated, mESC proliferation. GABA(A)R-specific ligands also induced morphological and gene expression changes indicating a differentiation shift. Our data suggest that the interplay between GABARs and downstream (coupled) effectors differentially modulates mESC proliferation/differentiation through selective activation of second messenger signaling cascades.-Schwirtlich, M., Emri, Z., Antal, K., Máté, Z., Katarova, Z., Szabó, G. GABA(A) and GABA(B) receptors of distinct properties affect oppositely the proliferation of mouse embryonic stem cells through synergistic elevation of intracellular Ca(2+).

Gamma-hydroxybutyrate does not maintain self-administration but induces conditioned place preference when injected in the ventral tegmental area.

Gamma-hydroxybutyric acid (GHB) is an endogenous brain substance that has diverse neuropharmacological actions, including rewarding properties in different animal species and in humans. As other drugs of abuse, GHB affects the firing of ventral tegmental neurons (VTA) in anaesthetized animals and hyperpolarizes dopaminergic neurons in VTA slices. However, no direct behavioural data on the effects of GHB applied in the VTA or in the target regions of its dopaminergic neurons, e.g. the nucleus accumbens (NAc), are available. Here, we investigated the effects of various doses of intravenous GHB in maintaining self-administration (from 0.001 to 10 mg/kg per infusion), and its ability to induce conditioned place preference (CPP) in rats when given orally (175-350 mg/kg) or injected directly either in the VTA or NAc (from 10 to 300 microg/0.5 microl per side). Our results indicate that while only 0.01 mg/kg per infusion GHB maintained self-administration, although not on every test day, 350 mg/kg GHB given orally induced CPP. CPP was also observed when GHB was injected in the VTA (30-100 microg/0.5 microl per side) but not in the NAc. Together with recent in-vitro findings, these results suggest that the rewarding properties of GHB mainly occur via disinhibition of VTA dopaminergic neurons.

Metabolic GHB precursor succinate binds to gamma-hydroxybutyrate receptors: characterization of human basal ganglia areas nucleus accumbens and globus pallidus.

Binding of the metabolic gamma-hydroxybutyrate (GHB) precursor succinate to NCS-382-sensitive [3H]GHB-labeled sites in crude synaptosomal or purified synaptic membrane fractions prepared from the human nucleus accumbens (NA), globus pallidus (GP) and rat forebrain has been shown. This site can be characterized by binding of ethyl hemisuccinate and gap-junction blockers, including carbenoxolone hemisuccinate and beta-GRA. There was no significant binding interaction between GABAB receptor ligands (CGP 55845, (R)-baclofen) and these [3H]GHB-labeled sites. GHB, NCS-382 and succinate binding profile of [3H]GHB-labeled sites in rat forebrain, human NA or GP synaptic membranes were similar. The synaptic fraction isolated from the rat forebrain was characterized by GHB binding inhibition constants: Ki,NCS-382 = 1.2 +/- 0.2 microM, Ki,GHB = 1.6 +/- 0.3 microM and Ki,SUCCINATE = 212 +/- 66 microM. In crude membranes containing mainly extrasynaptic membranes, distinct GHB and GABAB receptor sites were found in the NA. By contrast, extrasynaptic GABAB receptor sites of rat forebrain and GP were GHB- and succinate-sensitive, respectively. The heterogeneity of GABAB sites found in native membranes indicates GABAB receptor-dependent differences in GHB action. Based on these findings, we suggest that succinate (and possibly drugs available as succinate salt derivatives) can mimic some of the actions of GHB.

Suppression of neuronal network excitability and seizure-like events by 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine in juvenile rat hippocampus: involvement of a metabotropic glutamate receptor.

We present data on the antiepileptic potency of 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine (Q5) in juvenile (P9-13) rat hippocampal slices and in particular Q5's action mechanism and target. Q5 (200-500 microM), but not alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/Kainate receptor antagonists blocked low-[Mg2+]-induced seizure-like events (SLE) in the CA3 region. Q5 (100 microM) decreased Glu-induced [35S]guanosine 5'-O-(3-thiotriphosphate) binding enhancement in brain homogenates, without interaction with ionotropic Glu receptor sites and Glu transport. In voltage-clamped CA3 pyramidal cells, Q5 (500 microM) depressed activities of spontaneous excitatory and inhibitory postsynaptic currents without affecting miniature inhibitory currents. Metabotropic Glu receptor (mGluR) subtype antagonists affected network excitability dissimilarly. Intracellular Ca2+ ion transients induced by the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) were suppressed by Q5. Agreeing predictions obtained by modelling Q5 binding to different experimental conformations of mGlu1, Q5 was bound partially to an mGluR binding site in the presence of 1mM ACPD. Findings suggest the apparent involvement of a novel phenotype of action or a new mGluR subtype in the specific suppression of epileptiform activity by Q5 through the depression of network excitability.

Unravelling the brain targets of gamma-hydroxybutyric acid.

Gamma-hydroxybutyric acid (GHB) is a naturally occurring gamma-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).

Epileptogenesis and chronic seizures in a mouse model of temporal lobe epilepsy are associated with distinct EEG patterns and selective neurochemical alterations in the contralateral hippocampus.

Major aspects of temporal lobe epilepsy (TLE) can be reproduced in mice following a unilateral injection of kainic acid into the dorsal hippocampus. This treatment induces a non-convulsive status epilepticus and acute lesion of CA1, CA3c and hilar neurons, followed by a latent phase with ongoing ipsilateral neuronal degeneration. Spontaneous focal seizures mark the onset of the chronic phase. In striking contrast, the ventral hippocampus and the contralateral side remain structurally unaffected and seizure-free. In this study, functional and neurochemical alterations of the contralateral side were studied to find candidate mechanisms underlying the lack of a mirror focus in this model of TLE. A quantitative analysis of simultaneous, bilateral EEG recordings revealed a significant decrease of theta oscillations ipsilaterally during the latent phase and bilaterally during the chronic phase. Furthermore, the synchronization of bilateral activity, which is very high in control, was strongly reduced already during the latent phase and the decrease was independent of recurrent seizures. Immunohistochemical analysis performed in the contralateral hippocampus of kainate-treated mice revealed reduced calbindin-labeling of CA1 pyramidal cells; down-regulation of CCK-8 and up-regulation of NPY-labeling in mossy fibers; and a redistribution of galanin immunoreactivity. These changes collectively might limit neuronal excitability in CA1 and dentate gyrus, as well as glutamate release from mossy fiber terminals. Although these functional and neurochemical alterations might not be causally related, they likely reflect long-ranging network alterations underlying the independent evolution of the two hippocampal formations during the development of an epileptic focus in this model of TLE.

Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2.

GABA-mediated fast-hyperpolarizing inhibition depends on extrusion of chloride by the neuron-specific K-Cl cotransporter, KCC2. Here we show that sustained interictal-like activity in hippocampal slices downregulates KCC2 mRNA and protein expression in CA1 pyramidal neurons, which leads to a reduced capacity for neuronal Cl- extrusion. This effect is mediated by endogenous BDNF acting on tyrosine receptor kinase B (TrkB), with down-stream cascades involving both Shc/FRS-2 (src homology 2 domain containing transforming protein/FGF receptor substrate 2) and PLCgamma (phospholipase Cgamma)-cAMP response element-binding protein signaling. The plasmalemmal KCC2 has a very high rate of turnover, with a time frame that suggests a novel role for changes in KCC2 expression in diverse manifestations of neuronal plasticity. A downregulation of KCC2 may be a general early response involved in various kinds of neuronal trauma.

Binding crevice for TT-232 in a homology model of type 1 somatostatin receptor.

Somatostatin receptor type 1 was modelled based on the atomic structure of bovine rhodopsin. Possible ways of binding interaction between somatostatin receptor type 1 and TT-232, a cycloheptapeptide analogue of somatostatin with broad therapeutic potential, were analysed by molecular docking. The twelve TT-232 conformations, obtained by NMR measurements in H(2)O-D(2)O mixture, were similar, disclosing a consensus backbone conformation. Several residues interacting with TT-232, such as Val133, Asp137 (helix 3), Arg197 (helix 4), Phe287, Gln291, Asn294 (helix 6), Ser305, and Tyr313 (helix 7), were found. In accordance, in vitro binding experiments indicated high-affinity binding of TT-232 to (125)I labelled somatostatin sites in brain membranes. The single binding crevice obtained by docking may allow the design and discovery of new peptidomimetics of TT-232 in the future.

High-frequency synaptic input contributes to seizure initiation in the low-[Mg2+] model of epilepsy.

High-frequency field potential activity between 50 and 400 Hz occurs throughout seizure-like events recorded from the CA3 region of juvenile rat hippocampal slices under low-[Mg(2+)] condition. Another (400-800 Hz) component occurred mainly during preictal paroxysmal spiking and the onsets of seizure-like events (97%) and less frequently during tonic and clonic phases (38% and 70%, respectively). Short epochs of oscillations in this range were associated with fast negative field potential deflections at the start of field potential transients. Voltage-clamp recordings from putative CA3 pyramidal cells showed the occurrence of synaptic inputs in the same frequency range at the onset of seizure-like events and the beginning of preictal or clonic paroxysmal spikes, while the frequency of action potentials never reached that range. The amplitude of fast negative field potential deflection, the rise time of membrane potential or voltage-clamp current changes and the mean phase coherence were consistent with an increase of synchronization towards the onset of a seizure-like event. Their parallel changes indicate the involvement of both synaptic and nonsynaptic mechanisms in the synchronization of neuronal activity and the development of seizure-like events in the low-[Mg(2+)] model of epilepsy.

GABA(B) receptor antagonist CGP-36742 enhances somatostatin release in the rat hippocampus in vivo and in vitro.

Here, we show the modulation of somatostatin functions in the hippocampus by the orally active 'cognition enhancer' GABA(B) receptor antagonist, (3-aminopropyl)n-butylphosphinic acid (CGP-36742), both in vivo and in vitro. Using high-pressure liquid chromatography-coupled electrospray mass spectrometry, we measured a two-fold increase in the extracellular level of somatostatin to CGP-36742 application in the hippocampus of anaesthetised rats. The basal release of [125I]somatostatin in the synaptosomal fraction was increased by CGP-36742 in concentrations lower than 1 muM. Simultaneous measurement of [14C]Glu and [3H]gamma-aminobutyric-acid ([3H]GABA) showed that CGP-36742 increased their basal release. However, prior [125I]somatostatin application suppressed the increase in the basal release of [14C]Glu and induced a net decrease in the basal release of [3H]GABA. Somatostatin application had a similar effect. In slices, CGP-36742 increased the postsynaptic effect of somatostatin on CA1 pyramidal cells. These results suggest a pre- and postsynaptic functional 'cross-talk' between coexisting GABA(B) and somatostatin receptors in the rat hippocampus.

Increased inter-spike intervals and fast after-hyperpolarization of action potentials in rat hippocampal pyramidal cells accompanied with altered calbindin immunoreactivity 10-12 months after global forebrain ischemia.

In vivo electrophysiological recordings of CA1/CA2 pyramidal cells were performed 10-12 months after global forebrain ischemia (four-vessel occlusion, 15 mm) and were compared to levels of calbindin expression. Ischemic animals were subdivided in non-sclerotic ischemic (NSI) and sclerotic ischemic (SI) groups depending on the absence or presence of hippocampal sclerosis. A decreased excitability was observed in neurons from both groups, as shown by significant prolongation of inter-spike intervals (ISI) of evoked action potentials and by increased amplitude of fast after-hyperpolarization (fAHP). The ratio of calbindin-positive CA1/CA2 pyramidal cells decreased from 59% in control to 33% and 8% in NSI and SI animals, respectively. These results suggest that decreased excitability of CA1/CA2 pyramidal cells represents a protective mechanism against ischemia-induced neurodegeneration and might be related to decreased calbindin expression.