Cryo-EM structures of pannexin 1 and 3 reveal differences among pannexin isoforms.

Pannexins are single-membrane large-pore channels that release ions and ATP upon activation. Three isoforms of pannexins 1, 2, and 3, perform diverse cellular roles and differ in their pore lining residues. In this study, we report the cryo-EM structure of pannexin 3 at 3.9 Å and analyze its structural differences with pannexin isoforms 1 and 2. The pannexin 3 vestibule has two distinct chambers and a wider pore radius in comparison to pannexins 1 and 2. We further report two cryo-EM structures of pannexin 1, with pore substitutions W74R/R75D that mimic the pore lining residues of pannexin 2 and a germline mutant of pannexin 1, R217H at resolutions of 3.2 Å and 3.9 Å, respectively. Substitution of cationic residues in the vestibule of pannexin 1 results in reduced ATP interaction propensities to the channel. The germline mutant R217H in transmembrane helix 3 (TM3), leads to a partially constricted pore, reduced ATP interaction and weakened voltage sensitivity. The study compares the three pannexin isoform structures, the effects of substitutions of pore and vestibule-lining residues and allosteric effects of a pathological substitution on channel structure and function thereby enhancing our understanding of this vital group of ATP-release channels.

Intracellular activation of full-length human TREK-1 channel by hypoxia, high lactate, and low pH denotes polymodal integration by ischemic factors.

TREK-1, a two-pore domain potassium channel, responds to ischemic levels of intracellular lactate and acidic pH to provide neuroprotection. There are two splice variants of hTREK1: the shorter splice variant having a shorter N-terminus compared with the full-length hTREK1 with similar C-terminus sequence that is widely expressed in the brain. The shorter variant was reported to be irresponsive to hypoxia-a condition attributed to ischemia, which has put the neuroprotective role of hTREK-1 channel into question. Since interaction between N- and C-terminus of different ion channels shapes their gating, we re-examined the sensitivity of the full-length as well as the shorter hTREK-1 channel to intracellular hypoxia along with lactate. Single-channel data obtained from the excised inside-out patches of the full-length channel expressed in HEK293 cells indicated an increase in activity as opposed to a decrease in activity in the shorter isoform. However, both the isoforms showed an increase in activity under combined hypoxia, 20mM lactate, and low pH 6 condition, albeit with subtle differences in their individual actions, confirming the neuroprotective role played by hTREK-1 irrespective of the differences in the N-terminus among the splice variants. Furthermore, E321A mutant that disrupts the interaction of the C-terminus with the membrane showed a decrease in activity with hypoxia indicating the importance of the C-terminus in the hypoxic response of the full-length hTREK-1. We propose an increase in activity of both the splice variants of hTREK-1 in combined hypoxia, high lactate, and low pH conditions typically associated with ischemia provides neuroprotection.

Theta resonance and synaptic modulation scale activity patterns in the medial entorhinal cortex stellate cells.

Stellate cells (SCs) of the medial entorhinal cortex (MEC) are rich in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are known to effectively shape their activity patterns. The explanatory mechanisms, however, have remained elusive. One important but previously unassessed possibility is that HCN channels control the gain of synaptic inputs to these cells. Here, we test this possibility in rat brain slices, while subjecting SCs to a stochastic synaptic bombardment using the dynamic clamp. We show that in the presence of synaptic noise, HCN channels mainly exert their influence by increasing the relative signal gain in the theta frequency through the theta modulation of stochastic synaptic inputs. This subthreshold synaptic modulation then translates into a spiking resonance, which steepens with excitation in the presence of HCN channels. We present here a systematic assessment of synaptic theta modulation and trace its implications to the suprathreshold control of firing rate motifs. Such analysis was yet lacking in the SC literature. Furthermore, we assess the impact of noise statistics on this gain modulation and indicate possible mechanisms for the emergence of membrane theta oscillations and synaptic ramps, as observed in vivo. We support the data with a computational model that further unveils a competing role of inhibition, suggesting important implications for MEC computations.

Lactate reduces epileptiform activity through HCA1 and GIRK channel activation in rat subicular neurons in an in vitro model.

OBJECTIVE: Much evidence suggests that the subiculum plays a significant role in the regulation of epileptic activity. Lactate acts as a neuroprotective agent against many conditions that cause brain damage. During epileptic seizures, lactate formation reaches up to ~6 mmol/L in the brain. We investigated the effect of lactate on subicular pyramidal neurons after induction of epileptiform activity using 4-aminopyridine (4-AP-0Mg2+ ) in an in vitro epilepsy model in rats. The signaling mechanism associated with the suppression of epileptiform discharges by lactate was also investigated. METHODS: We used patch clamp electrophysiology recordings on rat subicular neurons of acute hippocampal slices. Immunohistochemistry was used for demonstrating the expression of hydroxycarboxylic acid receptor 1 (HCA1) in the subiculum. RESULTS: Our study showed that application of 6 mmol/L lactate after induction of epileptiform activity reduced spike frequency (control 2.5 ± 1.23 Hz vs lactate 1.01 ± 0.91 Hz, P = .049) and hyperpolarized the subicular neurons (control -51.8 ± 1.9 mV vs lactate -57.2 ± 3.56 mV, P = .002) in whole cell patch-clamp experiments. After confirming the expression of HCA1 in subicular neurons, we demonstrated that lactate-mediated effect occurs via HCA1 by using its specific agonist. All values are mean ±SD. Electrophysiological recordings revealed the involvement of Gßγ and intracellular cAMP in the lactate-induced effect. Furthermore, current-clamp and voltage-clamp experiments showed that the G protein-coupled inwardly rectifying potassium (GIRK) channel blocker tertiapin-Q, negated the lactate-induced inhibitory effect, which confirmed that lactate application results in outward GIRK current. SIGNIFICANCE: Our finding points toward the potential role of lactate as an anticonvulsant by showing lactate-induced suppression of epileptiform activity in subicular neurons. The study gives a different insight by suggesting importance of endogenous metabolite and associated signaling factors, which can aid in improving the present therapeutic approach for treating epilepsy.

High-speed waveguide integrated silicon photodetector on a SiN-SOI platform for short reach datacom.

We present a waveguide integrated high-speed Si photodetector integrated with a silicon nitride (SiN) waveguide on an silicon-on-insulator (SOI) platform for short reach data communication in a 850 nm wavelength band. We demonstrate a waveguide couple Si pin photodetector responsivity of 0.44 A/W at 25 V bias. The frequency response of the photodetector is evaluated by the coupling of a femtosecond laser source through an SiN grating coupler of the integrated photodetector. We estimate a 3 dB bandwidth of 14 GHz at 20 V bias which, to the best of our knowledge, is the highest reported bandwidth for a waveguide integrated Si photodetector. We also present detailed optoelectronic DC and AC characterization of the fabricated devices. The demonstrated integrated photodetector could enable an integrated solution for scaling of short reach data communication and connectivity.

Heterogeneous network dynamics in an excitatory-inhibitory network model by distinct intrinsic mechanisms in the fast spiking interneurons.

Fast spiking interneurons (FSINs) have an important role in neuronal network dynamics. Although plasticity of synaptic properties is known to affect network synchrony, the role of plasticity of FSINs' intrinsic excitability on network dynamics remain elusive. Using computational approaches in an excitatory-FSIN model network (EI) based on previously established hippocampal neuronal models we show that altered FSIN intrinsic excitability robustly affects the coherence and frequency of network firing monotonically in the connected excitatory network. Surprisingly, the effect of FSIN excitability was dependent on the mechanisms associated with changes in intrinsic excitability rather than on the direction of the change. Decreasing FSIN excitability by decreasing the membrane specific resistance (Rm), increasing peak HCN conductance (gihbar) increased the excitatory network coherence while increased peak delayed potassium conductance (gKDbar) decreased the coherence. However, the perturbations affected the excitatory network frequency in a similar manner. Further, in an isolated FSIN all-to-all network (II), decreasing FSIN excitability caused significant decrease in the network steady-state frequency due to any of the alterations. However, II network coherence remained unaltered with change in FSIN Rm but increased with higher gihbar and lower gKDbar. Interestingly, decreased FSIN Rin could partially rescue the decreasing EI network coherence with increasing gKDbar. The phenomenon of FSIN Rm, gihbar and gKDbar dependent EI network coherence alterations was robust for different proportions of plastic FSINs. Our results indicate that plasticity of intrinsic excitability in FSINs can regulate network dynamics and thus serve as an important network strategy during different physiological states.

Random Neuronal Networks show homeostatic regulation of global activity while showing persistent changes in specific connectivity paths to theta burst stimuli.

Learning in neuronal networks based on Hebbian principle has been shown to lead to destabilizing effects. Mechanisms have been identified that maintain homeostasis in such networks. However, the way in which these two opposing forces operate to support learning while maintaining stability is an active area of research. In this study, using neuronal networks grown on multi electrode arrays, we show that theta burst stimuli lead to persistent changes in functional connectivity along specific paths while the network maintains a global homeostasis. Simultaneous observations of spontaneous activity and stimulus evoked responses over several hours with theta burst training stimuli shows that global activity of the network quantified from spontaneous activity, which is disturbed due to theta burst stimuli is restored by homeostatic mechanisms while stimulus evoked changes in specific connectivity paths retain a memory trace of the training.

17ß-estradiol potentiates TREK1 channel activity through G protein-coupled estrogen receptor.

TWIK-related potassium channel 1 (TREK1), a two-pore domain potassium channel, is modulated by various hormones and neurotransmitters by activation of membrane receptor - coupled second messengers. 17ß-estradiol is a neuromodulator capable of regulating several cellular processes including the activity of ion channels, in a rapid and non-genomic manner. The G protein-coupled estrogen receptor (GPER) is known to facilitate rapid actions of 17ß-estradiol, though its role in modulation of ion channels is not widely explored. Several studies have shown both TREK1 and 17ß-estradiol to be neuromodulatory but the interaction between them is not known. In the present study, using single channel cell-attached patch clamp electrophysiology in HEK293 cells, we show that 17ß-estradiol increases the activity of hTREK1 channel by acting through hGPER and increasing the channel opening probability within minutes. The potentiation induced by 17ß-estradiol is pertussis toxin - sensitive involving action of Gßγ subunits while the inhibitory effect of cAMP-PKA pathway on TREK1 is reduced. Protein phosphatases were also found to be important for the action of 17ß-estradiol, which in concert with reduced activity of PKA, may alter the phosphorylation state of the channel and thus increase channel activity. Mutational studies revealed the serines at positions 315 and 348 in the C-terminal domain of hTREK1 to be the target sites for dephosphorylation induced by 17ß-estradiol action through hGPER. Elucidation of the pathway for the potentiating action of 17ß-estradiol via hGPER on hTREK1 channel activity will help us understand better one of the many possible neuroprotective mechanisms of 17ß-estradiol and hTREK1 channel.

Random neuronal ensembles can inherently do context dependent coarse conjunctive encoding of input stimulus without any specific training.

Conjunctive encoding of inputs has been hypothesized to be a key feature in the computational capabilities of the brain. This has been inferred based on behavioral studies and electrophysiological recording from animals. In this report, we show that random neuronal ensembles grown on multi-electrode array perform a coarse-conjunctive encoding for a sequence of inputs with the first input setting the context. Such an encoding scheme creates similar yet unique population codes at the output of the ensemble, for related input sequences, which can then be decoded via a simple perceptron and hence a single STDP neuron layer. The random neuronal ensembles allow for pattern generalization and novel sequence classification without needing any specific learning or training of the ensemble. Such a representation of the inputs as population codes of neuronal ensemble outputs, has inherent redundancy and is suitable for further decoding via even probabilistic/random connections to subsequent neuronal layers. We reproduce this behavior in a mathematical model to show that a random neuronal network with a mix of excitatory and inhibitory neurons and sufficient connectivity creates similar coarse-conjunctive encoding of input sequences.

l-Lactate mediates neuroprotection against ischaemia by increasing TREK1 channel expression in rat hippocampal astrocytes in vitro.

Brain ischaemia is a highly debilitating condition where shortage of oxygen and glucose leads to profuse cell death. Lactate is a neuroprotective metabolite whose concentrations increase up to 15-30 mmol/L during ischaemia and TREK1 is a neuroprotective potassium channel which is upregulated during ischaemia. The aim of this study was to investigate the effect of l-lactate on TREK1 expression and to evaluate the role of l-lactate-TREK1 interaction in conferring neuroprotection in ischaemia-prone hippocampus. We show that 15-30 mmol/L l-lactate increases functional TREK1 protein expression by 1.5-3-fold in hippocampal astrocytes using immunostaining and electrophysiology. Studies with transcription blocker actinomycin-D and quantitative PCR indicate that the increase in TREK1 expression is due to enhanced TREK1 mRNA transcription. We further report that l-lactate-mediated increase in TREK1 expression is via protein kinase A (PKA)-dependent pathway. This is the first report of an ischaemic metabolite affecting functional expression of an ion channel. Our studies in an in vitro model of ischaemia using oxygen glucose deprivation show that 30 mmol/L l-lactate fails to reduce cell death in rat hippocampal slices treated with TREK1 blockers, PKA inhibitors and gliotoxin. The above effects were specific to l-lactate as pyruvate failed to increase TREK1 expression and reduce cell death. l-Lactate-induced TREK1 upregulation is a novel finding of physiological significance as TREK1 channels contribute to neuroprotection by enhancing potassium buffering and glutamate clearance capacity of astrocytes. We propose that l-lactate promotes neuronal survival in hippocampus by increasing TREK1 channel expression via PKA pathway in astrocytes during ischaemia. Insufficient blood supply to the brain leads to cerebral ischaemia and increase in extracellular lactate concentrations. We incubated hippocampal astrocytes in lactate and observed increase in TREK1 channel expression via protein kinase A (PKA). Inhibition of TREK1, PKA and metabolic impairment of astrocytes prevented lactate from reducing cell death in ischaemic hippocampus. This pathway serves as an alternate mechanism of neuroprotection. Cover image for this issue: doi: 10.1111/jnc.13326.

Lactate modulates the intracellular pH sensitivity of human TREK1 channels.

Tissue acidosis and high lactate concentrations are associated with cerebral ischaemia. The degree of acidosis is dependent on circulating glucose concentration, hyperglycaemia being associated with increased acidosis. Among other agents, lactate and protons have been shown to activate the leak potassium channel; TREK1 (TWIK related potassium channel 1) from the intracellular side and its increased activity is implicated in tolerance towards ischaemic cell damage. In the present study, we show that ischaemic concentrations of lactate (30 mM) at pH 7.0 and 6.5, commonly observed during ischemia, cause robust potentiation of human TREK1 (hTREK1) activity at single-channel level in cell-free inside-out membrane patches, while 30 mM lactate at pH 6.0 to 5.5, commonly observed during hyperglycaemic ischemia, reduces hTREK1 channel activity significantly. The biphasic effect of 30 mM lactate (ischaemic concentrations) on modulation of hTREK1 by varying pH conditions is specific since basal concentrations of lactate (3 mM) and 30 mM pyruvate at pH 7.0 and 5.5 failed to show similar effect as lactate. Experiments with deletion and point mutants of hTREK1 channel suggest that lactate changes the pH modulation of hTREK1 by interacting differently with the histidine residue at 328th position (H328) above and below its pKa (∼6.0) in the intracellular carboxyl-terminal domain of TREK1. This lactate-induced pH modulation of hTREK1 is absent in C-terminal deletion mutant, CTDΔ100, and is similar in E321A-hTREK1 mutant as in wild-type hTREK1 suggesting that it is independent of pH-sensitive glutamate residue at 321st position. Such a differential pH-dependent effect of lactate on an ion channel function has not been reported earlier and has important implications in different stages of ischaemia.

Ischaemic concentrations of lactate increase TREK1 channel activity by interacting with a single histidine residue in the carboxy terminal domain.

KEY POINTS: The physiological metabolite, lactate and the two-pore domain leak potassium channel, TREK1 are known neuroprotectants against cerebral ischaemia. However, it is not known whether lactate interacts with TREK1 channel to provide neuroprotection. In this study we show that lactate increases TREK1 channel activity and hyperpolarizes CA1 stratum radiatum astrocytes in hippocampal slices. Lactate increases open probability and decreases longer close time of the human (h)TREK1 channel in a concentration dependent manner. Lactate interacts with histidine 328 (H328) in the carboxy terminal domain of hTREK1 channel to decrease its dwell time in the longer closed state. This interaction was dependent on the charge on H328. Lactate-insensitive mutant H328A hTREK1 showed pH sensitivity similar to wild-type hTREK1, indicating that the effect of lactate on hTREK1 is independent of pH change. A rise in lactate concentration and the leak potassium channel TREK1 have been independently associated with cerebral ischaemia. Recent literature suggests lactate to be neuroprotective and TREK1 knockout mice show an increased sensitivity to brain and spinal cord ischaemia; however, the connecting link between the two is missing. Therefore we hypothesized that lactate might interact with TREK1 channels. In the present study, we show that lactate at ischaemic concentrations (15-30 mm) at pH 7.4 increases TREK1 current in CA1 stratum radiatum astrocytes and causes membrane hyperpolarization. We confirm the intracellular action of lactate on TREK1 in hippocampal slices using monocarboxylate transporter blockers and at single channel level in cell-free inside-out membrane patches. The intracellular effect of lactate on TREK1 is specific since other monocarboxylates such as pyruvate and acetate at pH 7.4 failed to increase TREK1 current. Deletion and point mutation experiments suggest that lactate decreases the longer close dwell time incrementally with increase in lactate concentration by interacting with the histidine residue at position 328 (H328) in the carboxy terminal domain of the TREK1 channel. The interaction of lactate with H328 is dependent on the charge on the histidine residue since isosteric mutation of H328 to glutamine did not show an increase in TREK1 channel activity with lactate. This is the first demonstration of a direct effect of lactate on ion channel activity. The action of lactate on the TREK1 channel signifies a separate neuroprotective mechanism in ischaemia since it was found to be independent of the effect of acidic pH on channel activity.

Ascorbate protects neurons against oxidative stress: a Raman microspectroscopic study.

Oxidative stress due to excessive accumulation of reactive oxygen or nitrogen species in the brain as seen in certain neurodegenerative diseases can have deleterious effects on neurons. Hydrogen peroxide, endogenously generated in neurons under normal physiological conditions, can produce an excess of hydroxyl radical via a Fenton mediated mechanism. This may induce acute oxidative injury if not scavenged or removed effectively by antioxidants. There are several biochemical assay methods to estimate oxidative injury in cells; however, they do not provide information on the biochemical changes as the cells get damaged progressively under oxidative stress. Raman microspectroscopy offers the possibility of real time monitoring of the chemical composition of live cells undergoing oxidative stress under physiological conditions. In the present study, a hippocampal neuron coculture was used to observe the acute impact of hydroxyl radicals generated by hydrogen peroxide in the presence of Fe(2+) (Fenton reaction). Raman peaks related to nucleic acids (725, 782, 1092, 1320, 1340, 1420, and 1576 cm(-1)) showed time-dependent changes over the experimental period (60 min), indicating the breakdown of the phosphodiester backbone as well as nuclear bases. Interestingly, ascorbic acid (a potent antioxidant) when cotreated with Fenton reactants showed protection of cells as inferred from the Raman spectra, presumably by scavenging hydroxyl radicals. Little or no change in the Raman spectra was observed for untreated control cells and for cells exposed to Fe(2+) only, H2O2 only, and ascorbate only. A live-dead assay study also supported the current observations. Hence, Raman microspectroscopy has the potential to be an excellent noninvasive tool for early detection of oxidative stress that is seen in neurodegenerative diseases.

Calcium permeable AMPA receptor-dependent long lasting plasticity of intrinsic excitability in fast spiking interneurons of the dentate gyrus decreases inhibition in the granule cell layer.

The local fast-spiking interneurons (FSINs) are considered to be crucial for the generation, maintenance, and modulation of neuronal network oscillations especially in the gamma frequency band. Gamma frequency oscillations have been associated with different aspects of behavior. But the prolonged effects of gamma frequency synaptic activity on the FSINs remain elusive. Using whole cell current clamp patch recordings, we observed a sustained decrease of intrinsic excitability in the FSINs of the dentate gyrus (DG) following repetitive stimulations of the mossy fibers at 30 Hz (gamma bursts). Surprisingly, the granule cells (GCs) did not express intrinsic plastic changes upon similar synaptic excitation of their apical dendritic inputs. Interestingly, pairing the gamma bursts with membrane hyperpolarization accentuated the plasticity in FSINs following the induction protocol, while the plasticity attenuated following gamma bursts paired with membrane depolarization. Paired pulse ratio measurement of the synaptic responses did not show significant changes during the experiments. However, the induction protocols were accompanied with postsynaptic calcium rise in FSINs. Interestingly, the maximum and the minimum increase occurred during gamma bursts with membrane hyperpolarization and depolarization respectively. Including a selective blocker of calcium-permeable AMPA receptors (CP-AMPARs) in the bath; significantly attenuated the calcium rise and blocked the membrane potential dependence of the calcium rise in the FSINs, suggesting their involvement in the observed phenomenon. Chelation of intracellular calcium, blocking HCN channel conductance or blocking CP-AMPARs during the experiment forbade the long lasting expression of the plasticity. Simultaneous dual patch recordings from FSINs and synaptically connected putative GCs confirmed the decreased inhibition in the GCs accompanying the decreased intrinsic excitability in the FSINs. Experimentally constrained network simulations using NEURON predicted increased spiking in the GC owing to decreased input resistance in the FSIN. We hypothesize that the selective plasticity in the FSINs induced by local network activity may serve to increase information throughput into the downstream hippocampal subfields besides providing neuroprotection to the FSINs.

Input coding for neuro-electronic hybrid systems.

Liquid State Machines have been proposed as a framework to explore the computational properties of neuro-electronic hybrid systems (Maass et al., 2002). Here the neuronal culture implements a recurrent network and is followed by an array of linear discriminants implemented using perceptrons in electronics/software. Thus in this framework, it is desired that the outputs of the neuronal network, corresponding to different inputs, be linearly separable. Previous studies have demonstrated this by either using only a small set of input stimulus patterns to the culture (Hafizovic et al., 2007), large number of input electrodes (Dockendorf et al., 2009) or by using complex schemes to post-process the outputs of the neuronal culture prior to linear discriminance (Ortman et al., 2011). In this study we explore ways to temporally encode inputs into stimulus patterns using a small set of electrodes such that the neuronal culture's output can be directly decoded by simple linear discriminants based on perceptrons. We demonstrate that network can detect the timing and order of firing of inputs on multiple electrodes. Based on this, we demonstrate that the neuronal culture can be used as a kernel to transform inputs which are not linearly separable in a low dimensional space, into outputs in a high dimension where they are linearly separable. Thus simple linear discriminants can now be directly connected to outputs of the neuronal culture and allow for implementation of any function for such a hybrid system.

Depression biased non-Hebbian spike-timing-dependent synaptic plasticity in the rat subiculum.

The subiculum is a structure that forms a bridge between the hippocampus and the entorhinal cortex (EC), and plays a major role in the memory consolidation process. Here, we demonstrate spike-timing-dependent plasticity (STDP) at the proximal excitatory inputs on the subicular pyramidal neurons of juvenile rat. Causal (positive) pairing of a single EPSP with a single back-propagating action potential (bAP) after a time interval of 10 ms (+10 ms) failed to induce plasticity. However, increasing the number of bAPs in a burst to three, at two different frequencies of 50 Hz (bAP burst) and 150 Hz, induced long-term depression (LTD) after a time interval of +10 ms in both the regular-firing (RF), and the weak burst firing (WBF) neurons. The LTD amplitude decreased with increasing time interval between the EPSP and the bAP burst. Reversing the order of the pairing of the EPSP and the bAP burst induced LTP at a time interval of -10 ms. This finding is in contrast with reports at other synapses, wherein pre- before postsynaptic (causal) pairing induced LTP and vice versa. Our results reaffirm the earlier observations that the relative timing of the pre- and postsynaptic activities can lead to multiple types of plasticity profiles. The induction of timing-dependent LTD (t-LTD) was dependent on postsynaptic calcium change via NMDA receptors in the WBF neurons, while it was independent of postsynaptic calcium change, but required active L-type calcium channels in the RF neurons. Thus the mechanism of synaptic plasticity may vary within a hippocampal subfield depending on the postsynaptic neuron involved. This study also reports a novel mechanism of LTD induction, where L-type calcium channels are involved in a presynaptically induced synaptic plasticity. The findings may have strong implications in the memory consolidation process owing to the central role of the subiculum and LTD in this process.

Synconset waves and chains: spiking onsets in synchronous populations predict and are predicted by network structure.

Synfire waves are propagating spike packets in synfire chains, which are feedforward chains embedded in random networks. Although synfire waves have proved to be effective quantification for network activity with clear relations to network structure, their utilities are largely limited to feedforward networks with low background activity. To overcome these shortcomings, we describe a novel generalisation of synfire waves, and define 'synconset wave' as a cascade of first spikes within a synchronisation event. Synconset waves would occur in 'synconset chains', which are feedforward chains embedded in possibly heavily recurrent networks with heavy background activity. We probed the utility of synconset waves using simulation of single compartment neuron network models with biophysically realistic conductances, and demonstrated that the spread of synconset waves directly follows from the network connectivity matrix and is modulated by top-down inputs and the resultant oscillations. Such synconset profiles lend intuitive insights into network organisation in terms of connection probabilities between various network regions rather than an adjacency matrix. To test this intuition, we develop a Bayesian likelihood function that quantifies the probability that an observed synfire wave was caused by a given network. Further, we demonstrate it's utility in the inverse problem of identifying the network that caused a given synfire wave. This method was effective even in highly subsampled networks where only a small subset of neurons were accessible, thus showing it's utility in experimental estimation of connectomes in real neuronal-networks. Together, we propose synconset chains/waves as an effective framework for understanding the impact of network structure on function, and as a step towards developing physiology-driven network identification methods. Finally, as synconset chains extend the utilities of synfire chains to arbitrary networks, we suggest utilities of our framework to several aspects of network physiology including cell assemblies, population codes, and oscillatory synchrony.

Acute treatment with 17beta-estradiol attenuates astrocyte-astrocyte and astrocyte-neuron communication.

Astrocytes are now recognized as dynamic signaling elements in the brain. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. The ovarian steroid hormone, 17beta-estradiol, in addition to its rapid actions on neuronal electrical activity can rapidly alter astrocyte intracellular calcium concentration ([Ca2+]i) through a membrane-associated estrogen receptor. Using calcium imaging and electrophysiological techniques, we investigated the functional consequences of acute treatment with estradiol on astrocyte-astrocyte and astrocyte-neuron communication in mixed hippocampal cultures. Mechanical stimulation of an astrocyte evoked a [Ca2+]i rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a [Ca2+]i wave. Following acute treatment with estradiol, the amplitude of the [Ca2+]i elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the [Ca2+]i rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. Mechanical stimulation of astrocytes induced [Ca2+]i elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated [Ca2+]i rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in the presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings may indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol-mediated hormonal control.

Glutamate pretreatment affects Ca2+ signaling in processes of astrocyte pairs.

Simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca(2+) imaging, were used to study the spatial and temporal profiles of depolarization-induced changes in intracellular Ca(2+) ([Ca(2+)](i)) in the processes of cultured rat cortical astrocytes existing as pairs. Transient Ca(2+) changes locked to depolarization were observed as microdomains in the processes of the astrocyte pairs, and the responses were more pronounced in the adjoining astrocyte. Considering the functional significance of higher concentrations of glutamate observed in certain pathological conditions, Ca(2+) transients were recorded following pretreatment of cells with glutamate (500 microM for 20 min). This showed distance-dependent incremental scaling and attenuation in the presence of the metabotropic glutamate receptor (mGluR) antagonist, alpha-methyl(4-carboxy-phenyl) glycine (MCPG). Estimation of local Ca(2+) diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. Intracellular heparin introduced into the depolarized astrocyte did not affect the Ca(2+) transients in the heparin-loaded astrocyte but attenuated the [Ca(2+)](i) responses in the adjoining astrocyte, suggesting that inositol 1,4,5 triphosphate (IP(3)) may be the transfer signal. The uncoupling agent, 1-octanol, attenuated the [Ca(2+)](i) responses in both the control and glutamate pretreated astrocytes, indicating the role of gap junctional communication. Our studies indicate that individual astrocytes have distinct functional domains, and that the glutamate-induced alterations in Ca(2+) signaling involve a sequence of intra- and intercellular steps in which phospholipase C (PLC), IP(3), internal Ca(2+) stores, VGCC and gap junction channels appear to play an important role.

Kynurenate treatment of autaptic hippocampal microcultures affect localized voltage-dependent calcium diffusion in the dendrites.

It is not clear how different spatial compartments in the neuron are affected during epileptiform activity. In the present study we have examined the spatial and temporal profiles of depolarization induced changes in the intracellular Ca(2+) concentration in the dendrites of cultured autaptic hippocampal pyramidal neurons rendered epileptic experimentally by treatment with kynurenate (2 mM) and Mg(2+) (11.3 mM) in culture (treated neurons). This was examined with simultaneous somatic patch-pipette recording and Ca(2+) imaging experiments using the Ca(2+) indicator Oregon Green 488 BAPTA-1. Neurons stimulated by depolarization under whole-cell voltage clamp conditions revealed Ca(2+) entry at localized sites in the dendrites. Ca(2+) transients were observed even in the presence of NMDA and AMPA receptor antagonists suggesting that the opening of voltage gated calcium channels primarily triggered the local Ca(2+) changes. Peak Ca(2+) transients in the dendrites of treated neurons were larger compared to the signals recorded from the control neurons. Dendritic Ca(2+) transients in treated neurons showed a distance dependent scaling. Estimation of dendritic local Ca(2+) diffusion coefficients indicated higher values in the treated neurons and a higher availability of free Ca(2+). Simulation studies of Ca(2+) dynamics in these localized dendritic compartments indicate that local Ca(2+) buffering and removal mechanisms may be affected in treated neurons. Our studies indicate that small dendritic compartments are rendered more vulnerable to changes in intracellular Ca(2+) following induction of epileptiform activity. This can have important cellular consequences including local membrane excitability through mechanisms that remain to be elucidated.