Sujit Kumar Sikdar

Small-world network topology of hippocampal neuronal network is lost, in an in vitro glutamate injury model of epilepsy

Neuronal network topologies and connectivity patterns were explored in control and glutamate‐injured hippocampal neuronal networks, cultured on planar multielectrode arrays. Spontaneous activity was characterized by brief episodes of synchronous firing at many sites in the array (network bursts). During such assembly activity, maximum numbers of neurons are known to interact in the network. After brief glutamate exposure followed by recovery, neuronal networks became hypersynchronous and fired network bursts at higher frequency. Connectivity maps were constructed to understand how neurons communicate during a network burst. These maps were obtained by analysing the spike trains using cross‐covariance analysis and graph theory methods. Analysis of degree distribution, which is a measure of direct connections between electrodes in a neuronal network, showed exponential and Gaussian distributions in control and glutamate‐injured networks, respectively. Although both the networks showed random features, small‐world properties in these networks were different. These results suggest that functional two‐dimensional neuronal networks in vitro are not scale‐free. After brief exposure to glutamate, normal hippocampal neuronal networks became hyperexcitable and fired a larger number of network bursts with altered network topology. The small‐world network property was lost and this was accompanied by a change from an exponential to a Gaussian network.

Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line.

1. The rat brain type IIA Na+ channel alpha‐subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole‐cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell‐shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range ‐30 to + 40 mV, with a dominating fast component and a small slower component. 3. The steady‐state inactivation, h infinity, was related to potential by a Boltzmann distribution, underlying three states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range ‐70 to ‐120 mV were characterized by an initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin‐Huxley model, a three‐state scheme of the form O<‐‐>I1<‐‐>I2 was proposed, replacing the original two‐state scheme of the Hodgkin‐Huxley model, and the rate constants are reported. 6. The instantaneous current‐voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant‐field equation. 7. The time course of activation was described by an m chi model. However, the best‐fitted value of chi varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha m and beta m, were determined. Their functional dependence on the membrane potential was investigated.

Cholinergic role in monkey dorsolateral prefrontal cortex during bar-press feeding behavior

Cholinergic involvement in the neuronal activity of the dorsolateral (DL) prefrontal cortex in the monkey was investigated during bar-press feeding behavior. Iontophoretic application of ACh increased the firing rate of more than half of the cells through muscarinic receptors. Activity of ACh-sensitive cells did not correlate with any particular event during the feeding task. Continuous application of ACh markedly enhanced increases in response to events during the feeding task, and application of an ACh antagonist diminished response levels. This indicates that ACh release may occur during the feeding task and affect cortical cells to improve the signal-to-noise ratio of the excitatory input. Decreased responses to events during the feeding task diminished during ACh application. Driven discharges in the cells were evoked by stimulation of the basal nucleus of Meynert (BNM) where ACh containing cells are localized. This response was specifically blocked by iontophoretic application of atropine. In conclusion, cholinergic inputs arising from the BNM are distributed profusely among the cortical cells and modulate their excitability during bar-press feeding behavior.

Sodium Channel Modulating Activity in a δ-Conotoxin from an Indian Marine Snail

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG‐TCAFICIE‐NH2 has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C‐terminal amidation has been demonstrated by mass measurements on the C‐terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the δ‐conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage‐gated Na+ channel, stably expressed in Chinese hamster ovary cells. Unlike δ‐conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.

cAMP directly facilitates Ca‐induced exocytosis in bovine lactotrophs

We have used the whole cell patch clamp technique on single prolactin‐secreting bovine lactotrophs to measure plasma membrane capacitance (C m), an index of membrane surface area, under voltage‐clamp during cytosol dialysis with Ca and cAMP. cAMP increased the magnitude and rate of Ca‐induced exocytosis (C m increase) without affecting membrane conductance; however, cAMP had no detectable effect on C m when intracellular Ca was low. We thus report new evidence that cAMP can facilitate Ca‐induced secretion in a synergistic fashion, by acting directly on the secretory apparatus, independently of membrane conductance activation.

Effect of Fenvalerate, a pyrethroid insecticide on membrane fluidity

Fenvalerate is a commonly used pyrethroid insecticide, used to control a wide range of pests. We have studied its interaction with the membrane using fluorescence polarization and differential scanning calorimetry (DSC) techniques. Fenvalerate was found to decrease the DPH fluorescence polarization value of synaptosomal and microsomal membrane, implicating that it makes the membrane more fluid. At different concentrations of fenvalerate, the activation energy of the probe molecule in the membrane also changes revealed from the change in slope of the Arrhenius plot. At higher concentrations the insecticide slowly saturates the membrane. The effects of fenvalerate on model membrane were also studied with liposomes reconstituted with dipalmitoylphosphatidylcholine (DPPC). Fenvalerate decreased the phase transition temperature (Tm) of DPPC by 1.5 C degrees at 40 microM concentration, but there was no effect on the cooperativity of the transition as interpreted from the DSC thermogram. From the change in the thermogram profile with fenvalerate it has been interpreted that it localizes in the acyl chain region of the lipid, possibly between C10 and C16 region and weakens the acyl chain packing. Fenvalerate was also found to interact with DPPC liposomes containing cholesterol to fluidize it.

Characterization of contryphans from Conus loroisii and Conus amadis that target calcium channels

Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca(2+) channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP(D)WDPWC-NH(2) and Am975, GCO(D)WDPWC-NH(2) (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca(2+) channels. While Lo959 increases the Ca(2+) current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels.

Dual effects of G‐protein activation on Ca‐dependent exocytosis in bovine lactotrophs

The whole‐cell patch‐clamp technique was used to measure cell membrane capacitance (C m) to monitor exocytosis in single‐cultured bovine prolactin‐secreting cells (lactotrophs) of the anterior pituitary. The cells were dialyzed with solutions containing different concentrations of ionised Ca and non‐hydrolyzable GTP analogues (GTP‐γ‐S and GMP‐PNP) to activate G‐proteins. We have identified two distinct effects of G‐protein activation on Ca‐induced exocytosis: (i) the maximum C m increase due to intracellular Ca‐dependent exocytosis was diminished, suggesting an inhibitory role of G‐proteins close to the site of granule fusion, while (ii) the rate of C m increase (ΔC m/Δt) was facilitated, revealing conversely a stimulatory role of G‐proteins in the translocation of secretory granules to the fusion sites.

Modulation of the unitary exocytic event amplitude by cAMP in rat melanotrophs.

1 Secretory responses were measured in single rat pituitary melanotrophs as the relative increase in membrane capacitance (Cm) 8 min after the start of dialysis with solutions containing 0.45 μm Ca2+. In the added presence of cAMP (0.2 mM) in the patch pipette solution, capacitance responses increased 2‐ to 3‐fold in comparison with controls. 2 To study whether cAMP‐dependent mechanisms affect cytosolic calcium activity ([Ca2+]i), dibutyryl cyclic AMP (dbcAMP, 10 mM) was added to intact melanotrophs and [Ca2+]i was measured using fura‐2 AM. Addition of dbcAMP caused a transient reduction in [Ca2+]i to 82 ± 21 nM from a resting value of 100 ± 19 nM (mean ± s.e.m., n= 32, P < 0.002), indicating that the cAMP‐induced increase in secretory activity was not the result of cAMP acting to increase [Ca2+]i, which then increased secretory activity. 3 To investigate whether cAMP affects the secretory apparatus directly, the interaction of a single secretory granule with the plasmalemma was monitored by measuring discrete femtofarad steps in Cm. The signal‐to‐noise ratio of recordings was increased by pre‐incubating the cells with a hydrophobic anion, dipicrylamine. 4 Recordings of unitary exocytic events (discrete ‘on’ steps in Cm) showed that the amplitude of ‘on’ steps ‐ a parameter correlated to the size of exocytosing secretory granules ‐ increased from 4.2 ± 0.2 fF (n= 356) in controls to 7.9 ± 0.2 fF in the presence of cAMP (n= 329, P < 0.001), while the frequency of unitary exocytic events was similar in controls and in the presence of cAMP. 5 The results suggest that a cAMP‐dependent mechanism mediates the fusion of larger granules with the plasmalemma.

Selective inhibition of glucose-sensitive neurons in rat lateral hypothalamus by noxious stimuli and morphine.

On the basis of their responsiveness to electrophoretically applied glucose, neurons in the lateral hypothalamic area (LHA) have been characterized as either glucose sensitive or glucose nonsensitive. Glucose-sensitive neurons are important in feeding control (4, 36-38, 44, 54). The aim of this study was to increase understanding of the neurophysiological mechanisms involved in the disturbance of feeding by pain. Radiant heating of the scrotum, strong tail pinch, and immersion of the tail in hot water were used as noxious stimuli. In order to correlate the responses of LHA neurons to noxious inputs with possible local release of endogenous opiates, effects of electrophoretically applied morphine and naloxone were also tested. The effects of glucose, morphine, and noxious stimulation were studied in a total of 165 neurons recorded from 75 adult male urethane-chloralose-anesthetized rats. Of 52 neurons determined to be glucose sensitive, 36 (69%) were inhibited by both noxious stimulation and morphine. A majority of the glucose-nonsensitive neurons did not respond to either morphine or noxious stimulation (87/113, 74%). The relation of glucose sensitivity to inhibition by pain and/or morphine was statistically significant (Fishers exact probability test, P less than 0.01). Naloxone attenuated the inhibitory effects of both pain and morphine, thus suggesting mediation of both by the same neuronal mechanism. From this evidence we conclude that LHA glucose-sensitive neurons are involved in the suppression of feeding by noxious stimulation.