Effects of temperature and analyte application technique on neuron-based chemical sensing.

Results are presented which enhance the field of neuron-based sensing by providing insight on the effects of operating temperature and analyte application technique (pulse versus back-mixed) on sensing properties. In these studies, serotonin sensing attributes of giant visceral neurons VV1 and VV2 from the pond snail Lymnea stagnalis were measured. Experiments using a rapid fluid-exchange system reveal a concentration-dependent increase in maximum firing frequency similar to that reported earlier for a slow well-mixed application. With a rapid application, however, the maximum firing frequency is reached more quickly, and there is less cell-to-cell variability in both the maximum response and sensitivity. Given an application technique, an increase in temperature causes an increase in sensitivity and maximum firing frequency, as well as a decrease in the time required for the response to return to baseline following removal of the analyte. To provide insights on the kinetics of the serotonin-induced response, the effects of temperature and concentration on the rates of activation, recovery and desensitization were examined in detail. In general, it was found that an increase in temperature increases the rates of activation and desensitization, while the effects on recovery were not apparent. In addition, both the rates of activation and desensitization have a direct dependence on concentration while the rate of recovery has an inverse dependence.     

Expression and function of the neurotransmitter serotonin during development of the Helisoma nervous system

Exogenous serotonin has been shown to evoke a neuron-selective inhibition of neurite outgrowth and synaptogenesis in identified Helisoma neurons in vitro. We demonstrate here that serotonin is present in the embryonic nervous system of Helisoma and can act as a regulator of neuronal development in vivo. Serotonin-like immunoreactivity was first observed in neurons at an early stage of nervous system development (E20). Throughout embryogenesis, the number of serotonin-immunoreactive neurons increased in a stereotypic pattern that was unique for each type of ganglion. Strikingly, the number of serotonin-immunoreactive neurons continued to increase throughout adult life. Transient perturbation of endogenous serotonin levels during embryogenesis had profound effects on the development of specific identified neurons. Embryos treated with 5,7-dihydroxytryptamine and raised to maturity showed aberrations in neuronal morphology, neuronal dye coupling, and strength of electrical synaptic connections. These effects were restricted to neurons known to be sensitive to the growth-inhibitory effects of serotonin in vitro. These results support the hypothesis that neurotransmitters are an important class of regulatory factors during normal development of the nervous system.     

Photophysics of a neurotransmitter: ionization and spectroscopic properties of serotonin.

The neurotransmitter serotonin plays a modulatory role in the regulation of various cognitive and behavioral functions such as sleep, mood, pain, depression, anxiety, and learning by binding to a number of serotonin receptors present upon the cell surface. The spectroscopic properties of serotonin and their modulation with ionization state have been studied. Results show that serotonin fluorescence, as measured by its intensity, emission maximum, and lifetime, is pH dependent. These results are further supported by absorbance changes that show very similar pH dependence. Changes in fluorescence intensity and absorbance as a function of pH are consistent with a pK(a) of 10.4 +/- 0.2. The ligand-binding site for serotonin receptors is believed to be located in one of the transmembrane domains of the receptors. To develop a basis for monitoring the binding of serotonin to its receptors, its fluorescence in nonpolar media has been studied. No significant binding or partitioning of serotonin to membranes under physiological conditions was observed. Serotonin fluorescence in solvents of lower polarity is characterized by an enhancement in intensity and a blue shift in emission maximum, although the solvatochromism is much less pronounced than in tryptophan. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these results are relevant to future studies of serotonin and its binding to its receptors.     

The endogenous neurotransmitter, serotonin, modifies neuronal nitric oxide synthase activities

Serotonin, an important neurotransmitter, is colocalized with neuronal nitric oxide synthase (nNOS), a homodimeric enzyme which catalyzes the production of nitric oxide (NO(.-)) and/or oxygen species. As many interactions have been reported between the nitrergic and serotoninergic systems, we studied the effect of serotonin on nNOS activities. Our results reveal that nNOS is activated by serotonin as both NADPH consumption and oxyhemoglobin (OxyHb) oxidation were enhanced. The generation of L-citrulline from L-arginine (L-Arg) was not affected by serotonin in the range of 0-200 microM, suggesting an additional production of oxygen-derived species. But 5-hydroxytryptamine (5HT) induced the formation of both O and H(2)O(2) by nNOS, as evidenced by electron paramagnetic resonance (EPR) and by using specific spin traps. Overall, these results demonstrate that serotonin is able to activate nNOS, leading to the generation of reactive oxygen species (ROS) in addition to the NO(.-) production. Such a property must be considered in vivo as various nNOS-derived products mediate different signaling pathways.     

Evaluation of characteristic parameters for the neurotransmitter release mechanisms at the neuromuscular junction

The process of neurotransmitter release at the neuromuscular junction needs to be represented appropriately in modeling of the synaptic chemical transmission as a reaction-diffusion system. The release mechanisms of the expanding pore and the acceleration are analyzed by the computer simulation with respect to the effects of the characteristic parameters in the mechanisms on spontaneous generation of the miniature endplate current (MEPC), leading to the following evaluation. In the expanding pore mechanism the expanding rate of the pore more than 10 nm ms(-1) and the diffusion coefficient of acetylcholine in the synaptic cleft (D(c)) of about 1.0 x 10(-6) cm2 s(-1) yield the maximum amplitude, the rise time and the decay time constant of the MEPC in agreement with the empirical data. In the active release mechanism the 10-fold acceleration of the natural diffusion and a similar value of D(c) are required to suit for the empirical MEPC.     

Evaluation of the time course of neurotransmitter release from the measured PSC and MPSC

A method for obtaining delay histograms for the time course of neurotransmitter release is presented. The delay histogram is derived from the measured psc (or the sum of several psc's) and the mpsc (obtained experimentally or otherwise) by means of a simple, quick, mathematical procedure. The procedure may be automated for the greater part. No approximation of the mpsc shape is performed, and the method is applicable to all quantal contents. For low and medium quantal contents, the delay histograms obtained by the method are compared to those obtained by direct analysis. A reasonable agreement is achieved. An experiment of high quantal content, for which direct analysis is impossible, is then analysed using the new method. Difficulties which may arise when applying the procedure and methods to overcome them are discussed at length. Other methods are set forth in the Discussion.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Evaluation of the binding of serotonin by isolated CNS acidic lipids

Binding of serotonin by rat lipids was examined in an organic solvent-aqueous partition system. Only phospholipids and sulfatide were found to have appreciable activity: this technique was unsuitable for gangliosides due to their poor extractibility. Binding by phospholipid was abolished and that by sulfatide was greatly inhibited by increasing ionic strength in the aqueous phase. At an ionic strength of 0.3 M the apparent affinity of sulfatide for serotonin was about 3×10³ M. Both tryptamine and 5-methoxytryptamine were much more effective than serotonin in inhibiting the binding of radioactive serotonin, suggesting that the observed binding is simply a charge neutralization with little specificity. Binding of serotonin by mixed brain gangliosides was examined in an equilibrium dialysis system. Without adequate precautions, the chemical lability of serotonin was found to produce spurious data when binding was assessed by the distribution of radiolabel. Binding of serotonin by ganglioside was also greatly inhibited by increasing ionic strength: at 0.3 M an apparent affinity of about 10³ M was found. While dopamine did not inhibit the binding of radioactive serotonin, tryptamine, 5-methoxytryptamine, and serotonin were equally effective inhibitors.     

Association of different prediction methods for determination of the efficiency and selectivity on neuron-based sensors

A technique has been developed to determine the efficiency and the selectivity of a single neuron-based sensor in identifying the nature of the chemical agents in an unknown sample. This has been achieved by exploiting the unique electrical identifiers, also known as "signature patterns", generated by the neuronal cell membrane. These were generated based on the variations to the extracellular electrical activity, due to the effect of a broad range of chemical agents. We demonstrate the prediction capability of the sensor in identifying the nature of an unknown test sample from a combination of three chemical agents, namely, ethanol, pyrethroid, and hydrogen peroxide. This was achieved through a two-step process. The first step was experimentally achieved by in situ recording of the changes to the extracellular electrical activity from the sensing sites or the array of microelectrodes that form the platform for patterning neurons. Simultaneous optical characterization of the cell array during the sensing process was performed to identify the associated physiological changes. The second step was mathematical and was based on developing a library of signature patterns for a set of concentrations of the various combinations of the three chemical agents. Two variants of the nearest neighbor algorithm scheme - (a) partial distance search method, and (b) search tree method, were implemented for the accurate detection of all the components with varying concentrations in the test samples of unknown nature. This technique exhibits reliability in identification up to parts-per-billion (ppb) sensitivity. The capability of standardization of this technique for potential commercial applications is also discussed.     

Differences in the in vivo dynamics of neurotransmitter release and serotonin uptake after acute para-methoxyamphetamine and 3,4-methylenedioxymethamphetamine revealed by chronoamperometry

Illicit use of p-methoxyamphetamine (PMA) is rapidly increasing. However, little is known about the acute effects of PMA on neurotransmission in vivo. High-speed chronoamperometry was used to monitor neurotransmitter release and clearance in anesthetized rats after local application of PMA or 3,4-methylenedioxymethamphetamine (MDMA). In striatum, PMA caused less neurotransmitter release than MDMA. PMA-evoked release could be partially blocked by pre-treatment with a serotonin (5-HT) reuptake inhibitor, suggesting that evoked 5-HT release contributed to the electrochemical signal and was mediated by the 5-HT transporter (SERT). MDMA-evoked release was not blocked by a SERT inhibitor, suggesting that primarily DA was released. To study the effect of these amphetamines on clearance of 5-HT mediated specifically by the SERT, clearance of exogenously applied 5-HT was measured in the CA3 region of the hippocampus. In contrast to the striatum where 5-HT is cleared by both the SERT and the dopamine transporter (DAT), 5-HT is cleared primarily by the SERT in the CA3 region. This is also a region where neither PMA nor MDMA evoked release of neurotransmitter. The maximal inhibition of 5-HT clearance was greater after PMA than MDMA. These data demonstrate in vivo (1) brain region variability in the ability of PMA and MDMA to evoke release of neurotransmitter; (2) that clearance of 5-HT in the striatum is mediated by both the SERT and the DAT; (3) distinct differences in the amount and nature of neurotransmitter released in the striatum after local application of PMA and MDMA and (4) that PMA is a more efficacious inhibitor of 5-HT clearance in the hippocampus than MDMA. These fundamental differences may account for the more severe adverse reactions seen clinically after PMA, compared to MDMA.     

A conserved asparagine residue in transmembrane segment 1 (TM1) of serotonin transporter dictates chloride-coupled neurotransmitter transport

Na(+)- and Cl(-)-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl(-) coordination of human serotonin transport have been identified, the role of Cl(-) in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl(-) binding to concentrative neurotransmitter uptake.     

Brain neurotransmitter receptors after long-term haloperidol: dopamine, acetylcholine, serotonin, alpha-noradrenergic and naloxone receptors.

Since long-term neuroleptic therapy is known to alter brain dopaminergic sensitivity, we tested the effects of chronic haloperidol administration (10 mg/kg/day for over 3 weeks) on the amount of the dopamine receptors (using ³H-apomorphine and ³H-haloperidol) in various regions of the rat brain. To test whether the changes in dopamine receptors were selectively produced, we also assayed acetylcholine receptors (with ³H-quinuclidinyl benzilate or ³H-QNB), alpha-noradrenergic receptors (with ³H-WB-4101), ³H-serotonin receptors and ³H-naloxone receptors.The specific binding of ³H-haloperidol increased significantly by 34% in the striatum and by 45% in the mesolimbic region after long-term haloperidol. The specific binding of ³H-apomorphine also increased significantly by 77% in the striatum and 55% in the mesolimbic area. Although there was a small significant increase of 20% in specific ³H-serotonin binding in the striatum, no such increment occurred in the hippocampus or the cerebral cortex. No significantly different binding occurred for the other ³H-ligands in these brain regions except for a 13% increase in alpha-noradrenergic binding in the cerebral cortex. These results indicate that long-term haloperidol treatment produces rather selective increases in dopamine/neuroleptic receptors, without much change in 4 other types of receptors. Such relatively selective increments in these receptors may be the basis of dopaminergic supersensitivity (e.g. tardive dyskinesia) after long-term haloperidol.     

Constraining the connectivity of neuronal networks cultured on microelectrode arrays with microfluidic techniques: a step towards neuron-based functional chips

In vitro culture of small neuronal networks with pre-defined topological features is particularly desirable when the electrical activity of such assemblies can be monitored for long periods of time. Indeed, it is hoped that such networks, with pre-determined connectivity, will provide unique insights into the structure/function relationship of biological neural networks and their properties of self-organization. However, the experimental techniques that have been developed so far for that purpose have either failed to provide very long-term pattern definition and retention, or they have not shown potential for integration into more complex microfluidic devices. To address this problem, three-dimensional microfluidic systems in poly(dimethylsiloxane) (PDMS) were fabricated and used in conjunction with both custom-made and commercially available planar microelectrode arrays (pMEAs). Various types of primary neuronal cell cultures were established inside these systems. Extracellular electrical signals were successfully recorded from all types of cells placed inside the patterns, and this bioelectrical activity was present for several weeks. The advantage of this approach is that it can be further integrated with microfluidic devices and pMEAs to yield, for example, complex neuron-based biosensors or chips for pharmacological screening.     

Evaluation of anti-quorum sensing activity of silver nanowires

A menace of antimicrobial resistance with growing difficulties in eradicating clinical pathogens owing to the biofilm has prompted us to take up a facile aqueous-phase approach for the synthesis of silver nanowires (SNWs) by using ethylene glycol as a reducing agent and polyvinylpyrrolidone (PVP) as a capping agent. This synthesis is a reflux reaction seedless process. The obtained SNWs were about 200–250 nm in diameter and up to 3–4 μm in length. The SNWs were characterized by field emission scanning electron microscopy, transmission electron microscopy, UV–Vis spectroscopy, and X-Ray powder diffraction, and the chemical composition of the sample was examined by energy dispersive X-ray spectrum. The SNWs did not show an antibacterial activity against test organisms, Bacillus subtilis NCIM 2063 and Escherichia coli NCIM 2931; however, it showed a promising property of a quorum sensing-mediated inhibition of biofilm in Pseudomonas aeruginosa NCIM 2948 and violacein synthesis in Chromobacterium violaceum ATCC 12472, which is hitherto unattempted, by polyol approach.     

Evaluation of isotryptamine derivatives at 5-HT2 serotonin receptors

On the basis that meta-chlorophenylpiperazine (mCPP; 1) is a nonselective 5-HT_2C agonist, that benz-fused tryptamines (e.g., 5) display enhanced 5-HT₂ affinity, and that certain isotryptamines 3 reportedly bind with enhanced affinity and selectivity at 5-HT_2C receptors, we prepared and examined a series of isotryptamine-related analogues as potentially selective 5-HT_2C agonists. None of the compounds displayed selectivity for 5-HT_2C versus 5-HT_2A receptors. Detailed re-examination of a compound previously reported to display 100-fold 5-HT_2C selectivity [i.e., S(+)-5,6-difluoro-α-methylisotryptamine] revealed that its selectivity versus 5-HT_2A receptors was, at best, only 10-fold.