Beadex, the Drosophila LIM only protein, is required for the growth of the larval neuromuscular junction.

The appropriate growth of the neurons, accurate organization of their synapses, and successful neurotransmission are indispensable for sensorimotor activities. These processes are highly dynamic and tightly regulated. Extensive genetic, molecular, physiological, and behavioral studies have identified many molecular candidates and investigated their roles in various neuromuscular processes. In this paper, we show that Beadex (Bx), the Drosophila LIM only (LMO) protein, is required for motor activities and neuromuscular growth of Drosophila. The larvae bearing Bx7, a null allele of Bx, and the RNAi-mediated neuronal-specific knockdown of Bx show drastically reduced crawling behavior, a diminished synaptic span of the neuromuscular junctions (NMJ) and an increased spontaneous neuronal firing with altered motor patterns in the central pattern generators (CPGs). Microarray studies identified multiple targets of Beadex that are involved in different cellular and molecular pathways, including those associated with the cytoskeleton and mitochondria, that could be responsible for the observed neuromuscular defects. With genetic interaction studies, we further show that Highwire (Hiw), a negative regulator of synaptic growth at the NMJs, negatively regulates Bx, as the latter's deficiency was able to rescue the phenotype of the Hiw null mutant, HiwDN. Thus, our data indicates that Beadex functions downstream of Hiw to regulate the larval synaptic growth and physiology.

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.

Prolonged exposure to lactate causes TREK1 channel clustering in rat hippocampal astrocytes.

Increased concentrations of lactate (15-30 mM) are associated with and found to be neuroprotective in various brain pathophysiology. In our earlier studies we showed that high levels of lactate can increase TREK1 channel activity and expression within 1 h. TREK1 channels are two pore domain leak potassium ion channels that are upregulated during cerebral ischemia, epilepsy and other brain pathologies. They play a prominent neuroprotective role against excitotoxicity. Although it has been previously shown that chronic application of lactate (6 h) causes increased gene transcription and protein expression, we observe clustering of TREK1 channels that is dependent on time of exposure (3-6 h) and concentration of lactate (15-30 mM). Using immunofluorescence techniques and image analysis, we show that the clustering of TREK1 channels is dependent on the actin cytoskeletal network of the astrocytes. Clustering of TREK1 channels can augment astrocytic functions during pathophysiological conditions and have significant implications in lactate mediated neuroprotection.

Cholinergic receptor-independent modulation of intrinsic resonance in the rat subiculum neurons through inhibition of hyperpolarization-activated cyclic nucleotide-gated channels.

AIM: Acetylcholine release is vital in the pacing of theta rhythms in the hippocampus. The subiculum is the output region of the hippocampus with different neuronal subtypes that generate theta oscillations during arousal and rapid eye movement sleep. The combination of intrinsic resonance in the hippocampal neurons and the periodic excitation of hippocampal excitatory and inhibitory neurons by cholinergic pathway drives theta oscillations. However, the acetylcholine mediated effect on intrinsic subthreshold resonance generating hyperpolarization-activated cyclic nucleotide-gated current, Ih of subicular neurons is unexplored. We studied the acetylcholine receptor-independent effect of cholinergic agents on the intrinsic properties of subiculum principal neurons and the underlying mechanism. METHODS: We bath perfused acetylcholine or nicotine on rat brain slices in the presence of synaptic blockers. The physiological effect was studied by cholinergic fibres stimulation and electrophysiological recordings under whole-cell mode of subiculum neurons using septohippocampal sections. RESULTS: Exogenously applied acetylcholine in the presence of atropine affected two groups of subicular neurons differently. Acetylcholine reduced the resonance frequency and Ih in bursting neurons, whereas these properties were unaffected in regular firing neurons. Subsequently, the endogenously released acetylcholine by stimulation showed a selective suppressive effect on Ih , sag, and resonance in burst firing among the two excitatory neurons. Nicotine suppressed the Ih amplitude in burst firing neurons, which was evident by decreased sag amplitude and resonance frequency and increased excitability. CONCLUSION: Our study suggests cell type-specific acetylcholine receptor-independent shift in resonance frequency by partially inhibiting HCN current during high cholinergic inputs.

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.

Compact ring resonator enhanced silicon metal-semiconductor-metal photodetector in SiN-on-SOI platform.

We present a compact on-chip resonator enhanced silicon metal-semiconductor-metal (MSM) photodetector in 850 nm wavelength band for communication and lab-on-chip bio-sensing applications. We report the highest responsivity of 0.81 A/W for a 5 µm long device. High responsivity is achieved by integrating the detector in a silicon nitride ring resonator. The resonance offers 100X responsivity improvement over a single-pass photodetector due to cavity enhancement. We also present a detailed study of the high-speed response of the cavity and single-pass detector. We report an electro-optic bandwidth of 7.5 GHz measured using a femtosecond optical excitation. To the best of our knowledge, we report for the first time silicon nitride resonator integrated Si-MSM detector in SiN-SOI platform.

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.

Efficient neural differentiation of mouse pluripotent stem cells in a serum-free medium and development of a novel strategy for enrichment of neural cells.

Pluripotent stem cells (PSCs) offer an excellent model to study neural development and function. Although various protocols have been developed to direct the differentiation of PSCs into desired neural cell types, many of them suffer from limitations including low efficiency, long duration of culture, and the use of expensive, labile, and undefined growth supplements. In this study, we achieved efficient differentiation of mouse PSCs to neural lineage, in the absence of exogenous molecules, by employing a serum-free culture medium containing knockout serum replacement (KSR). Embryoid bodies (EBs) cultured in this medium predominantly produced neural cells which included neural progenitors (15-18%), immature neurons (8-24%), mature neurons (10-26%), astrocytes (27-61%), and oligodendrocytes (∼1%). Different neuronal subtypes including glutamatergic, GABAergic, cholinergic, serotonergic, and dopaminergic neurons were generated. Importantly, neurons generated in the KSR medium were electrically active. Further, the EB scooping strategy, involving the removal of the EB core region from the peripheral EB outgrowth, resulted in the enrichment of PSC-derived neural cells. Taken together, this study provides the evidence that the KSR medium is ideal for the rapid and efficient generation of neural cells, including functional neurons, from PSCs without the requirement of any other additional molecule.

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.

Early postnatal exposure to lithium in vitro induces changes in AMPAR mEPSCs and vesicular recycling at hippocampal glutamatergic synapses.

Lithium is an effective mood stabilizer but its use is associated with many side effects. Electrophysiological recordings of miniature excitatory postsynaptic currents (mEPSCs) mediated by glutamate receptor AMPA-subtype (AMPARs) in hippocampal pyramidal neurons revealed that CLi (therapeutic concentration of 1 mM lithium, from days in vitro 4-10) decreased the mean amplitude and mean rectification index (RI) of AMPAR mEPSCs. Lowered mean RI indicate that contribution of Ca2+ -permeable AMPARs in synaptic events is higher in CLi neurons (supported by experiments sensitive to Ca2+ -permeable AMPAR modulation). Co-inhibiting PKA, GSK-3 beta and glutamate reuptake was necessary to bring about changes in AMPAR mEPSCs similar to that seen in CLi neurons. FM1-43 experiments revealed that recycling pool size was affected in CLi cultures. Results from minimum loading, chlorpromazine treatment and hyperosmotic treatment experiments indicate that endocytosis in CLi is affected while not much difference is seen in modes of exocytosis. CLi cultures did not show the high KCl associated presynaptic potentiation observed in control cultures. This study, by calling attention to long-term lithium-exposure-induced synaptic changes, might have implications in understanding the side effects such as CNS complications occurring in perinatally exposed babies and cognitive dulling seen in patients on lithium treatment.

A Disulfide Stabilized ß-Sandwich Defines the Structure of a New Cysteine Framework M-Superfamily Conotoxin.

The structure of a new cysteine framework (-C-CC-C-C-C-) "M"-superfamily conotoxin, Mo3964, shows it to have a ß-sandwich structure that is stabilized by inter-sheet cross disulfide bonds. Mo3964 decreases outward K(+) currents in rat dorsal root ganglion neurons and increases the reversal potential of the NaV1.2 channels. The structure of Mo3964 (PDB ID: 2MW7 ) is constructed from the disulfide connectivity pattern, i.e., 1-3, 2-5, and 4-6, that is hitherto undescribed for the "M"-superfamily conotoxins. The tertiary structural fold has not been described for any of the known conus peptides. NOE (549), dihedral angle (84), and hydrogen bond (28) restraints, obtained by measurement of (h3)JNC' scalar couplings, were used as input for structure calculation. The ensemble of structures showed a backbone root mean square deviation of 0.68 ± 0.18 Å, with 87% and 13% of the backbone dihedral (ϕ, ψ) angles lying in the most favored and additional allowed regions of the Ramachandran map. The conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules.