Combined antenna and localized plasmon resonance in Raman scattering from random arrays of silver-coated, vertically aligned multiwalled carbon nanotubes.

The electric field enhancement associated with detailed structure within novel optical antenna nanostructures is modeled using the surface integral equation technique in the context of surface-enhanced Raman scattering (SERS). The antennae comprise random arrays of vertically aligned, multiwalled carbon nanotubes dressed with highly granular Ag. Different types of "hot-spot" underpinning the SERS are identified, but contrasting characteristics are revealed. Those at the outer edges of the Ag grains are antenna driven with field enhancement amplified in antenna antinodes while intergrain hotspots are largely independent of antenna activity. Hot-spots between the tops of antennae leaning towards each other also appear to benefit from antenna amplification.

Targeting of serotonin 1A receptors to dopaminergic neurons within the parabrachial subdivision of the ventral tegmental area in rat brain.

Serotonin (5-hydroxytryptamine [5-HT]) modulates dopamine-related cognitive functions and motor activity through activation of selective receptor subtypes including 5-HT1A. Potential targets for these 5-HT1A-mediated actions of 5-HT include mesocortical and mesolimbic dopaminergic neurons having partially segregated distribution in the parabrachial and paranigral subdivisions of the ventral tegmental area (VTA), respectively. We therefore examined the ultrastructural immunocytochemical localization of the 5-HT1A receptor in the parabrachial (VTApb) and paranigral (VTApn) subdivisions of rat VTA, to determine 1) the functional sites for receptor activation, and 2) the cellular associations between this receptor and dopaminergic neurons identified by their tyrosine hydroxylase (TH) content. In each region, 5-HT1A immunoreactivity was mainly observed in somatodendritic profiles, but it was also present in small unmyelinated axons and in a few axon terminals and glia, suggesting a role for 5-HT1A receptors in presynaptic and glial functions, as well as postsynaptic neuronal activation, in VTA. In somatodendritic profiles, 5-HT1A gold particles were mainly localized to tubulovesicles presumed to be smooth endoplasmic reticulum. In addition, however, in distal dendrites receiving multiple inputs the receptor was targeted to selective postsynaptic junctions, or more randomly distributed on nonsynaptic portions of the plasma membrane. Of the 5-HT1A-labeled dendrites, 64% in VTApb and 44% in VTApn contained TH. These findings suggest a reserve of cytoplasmic 5-HT1A receptors that are mobilized to functional postsynaptic sites on the plasma membrane by afferent input to distal dendrites in the VTA. They also indicate that 5-HT1A activation may affect a larger population of dopaminergic neurons in VTApb compared with VTApn, thus having a potentially greater impact on cognitive functions modulated by mesocortical dopaminergic neurons.

Ultrastructural localization of the serotonin 2A receptor in dopaminergic neurons in the ventral tegmental area.

Serotonin (5-hydroxytryptamine, 5-HT) 2A receptor antagonists are clinically effective antipsychotics that may differentially target mesocortical and mesolimbic dopaminergic neurons having partially segregated distribution in the parabrachial (PB) and paranigral (PN) ventral tegmental area (VTA). We examined the ultrastructural immunocytochemical localization of the 5-HT2A receptor in these subdivisions of rat VTA, to determine (1) the functional sites for receptor activation, and (2) cellular associations between the receptor and dopaminergic neurons identified by their content of tyrosine hydroxylase (TH). The mean area density of neuronal profiles containing 5-HT2A receptor labeling was not significantly different in the PB and PN VTA. In each region approximately 44% of the 5-HT2A labeled profiles were dendrites while the remainder were mainly axons. Dendritic 5-HT2A-immunoreactivity was often localized to membranous cytoplasmic organelles resembling smooth endoplasmic reticulum, and to more rarely to segments of the plasma membrane beneath contacts from unlabeled axon terminals. 5-HT2A labeling was also seen within the cytoplasm of a few axon initial segments and many small unmyelinated axons. Approximately 40% of the 5-HT2A-labeled dendritic profiles contained TH in either PB or PN VTA. Our results suggest that 5-HT2A receptors in VTA are largely cytoplasmic and play an equally important role in modulating dopaminergic neurons in PB and PN VTA. These results also implicate 5-HT2A receptors in the postsynaptic activation of non-dopaminergic neurons and possibly the presynaptic release from terminals of axons originating in, or passing through, these regions.

N-methyl-D-aspartate (NMDA) receptors in the ventral tegmental area: subcellular distribution and colocalization with 5-hydroxytryptamine(2A) receptors.

Glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype have been implicated in behavioral sensitization to psychostimulants and in psychotic behaviors involving excitation of ventral tegmental area (VTA) dopaminergic neurons. Antagonists of serotonin (5-hydroxytryptamine, 5-HT) receptors of the 5-HT(2A) subtype are potent antipsychotics that attenuate these NMDA-evoked responses. We examined the electron microscopic immunocytochemical localization of antisera against the NMDA R1 subunit (NMDAR1) and 5-HT(2A) receptors to determine potential sites for their dual activation in the rat paranigral and parabrachial VTA subdivisions that are distinguished, in part, by their respective striatolimbic and cortical projections. In both regions, NMDAR1 immunoreactivity was localized mainly to the cytoplasm of somata and dendrites, and was only occasionally seen near or within excitatory-type asymmetric synapses. Many of the NMDAR1-labeled somata and dendrites also expressed 5-HT(2A) receptors, having a similar, but largely non-overlapping, neuronal distribution. The mean area density of NMDAR1 and dually labeled dendritic profiles was significantly greater in the paranigral than in the parabrachial VTA. NMDAR1 was also present in small axons showing a similar regional difference in area density. No regional difference in area density was seen in dendrites or small axons containing only 5-HT(2A) receptors. Our results indicate that NMDA and 5-HT(2A) receptors in the VTA are transiently expressed on synaptic plasma membranes of single neurons showing widespread cytoplasmic distributions of each of the receptors. They also suggest a major role for NMDA receptors in modulating the output of paranigral neurons and the release of transmitters from axons passing through this region.

Effects of medial prefrontal cortical injections of GABA receptor agonists and antagonists on the local and nucleus accumbens dopamine responses to stress.

Stress stimulates dopamine (DA) release in nucleus accumbens (NAcc) but will do so more strongly in medial prefrontal cortex (PFC). Evidence indicates, however, that the cortical DA response to stress acts to dampen the concurrent increase in NAcc DA release. In the present study, we used voltammetry to investigate the role of PFC GABA in regulating the NAcc DA response to stress. The results of Experiment 1 show that the NAcc stress response is inhibited following bilateral cortical microinjections of baclofen (GABAB receptor agonist). While phaclofen (GABAB receptor antagonist) blocked the effect of baclofen, it had no significant effect of its own. Intra-PFC injections of muscimol (GABAA receptor agonist) and bicuculline (GABAA receptor antagonist) had no effect on the DA stress response in NAcc. In Experiment 2, we explored the possibility that GABA influences the NAcc DA stress response indirectly by modulating stress-induced DA release in PFC. None of the drugs tested had an effect on the PFC stress response at a dose (1 nmol) that produced reliable effects on the NAcc stress response. At an order of magnitude higher dose, however, locally applied phaclofen and muscimol enhanced and attenuated, respectively, the DA stress response in PFC. These results were validated in Experiment 3 by showing that intra-PFC injections of GBR-12395 (DA uptake blocker) and quinpirole (D2/D3 receptor agonist) dose-dependently enhanced and inhibited, respectively, the local DA stress response. Together, these findings indicate that increased GABA transmission in PFC exerts an inhibitory influence on the NAcc DA response to stress, and that this action is mediated primarily but not exclusively by GABAB receptors which may be located both on cortical output neurons and on DA terminals.

NMDA receptors in nucleus accumbens modulate stress-induced dopamine release in nucleus accumbens and ventral tegmental area.

Converging evidence suggests that dopamine (DA) transmission in nucleus accumbens (NAcc) is modulated locally by an excitatory amino acid (EAA)-containing input possibly originating in medial prefrontal cortex (PFC). In the present study, we examined the effects of intra-NAcc administration of EAA receptor antagonists on stress-induced increases of NAcc DA levels and of dendritically released DA in the ventral tegmental area (VTA). Local injection of the NMDA receptor antagonist-AP-5 (0.05, 0.5, and 5.0 nmoles)-dose-dependently potentiated increases in NAcc DA levels elicited by 15 min of restraint stress. In contrast, local application of equivalent doses of the kainate/AMPA receptor antagonist-DNQX-failed to alter the NAcc DA stress response reliably. In a separate experiment, we found that intra-NAcc injection of AP-5 also potentiated stress-induced increases in VTA DA levels. These results indicate that EAAs acting at NMDA receptors in NAcc can modulate stress-induced DA release in this region. Our data indicate, however, that this action exerts an inhibitory influence on the NAcc DA stress response, suggesting that the relevant population of NMDA receptors are not located on NAcc DA terminals. The fact that intra-NAcc AP-5 injections also potentiated the DA stress response in VTA suggests instead an action mediated by NMDA receptors located on NAcc neurons that feedback, directly or indirectly, to cell bodies of the mesocorticolimbic DA system.

Medial prefrontal cortical D1 receptor modulation of the meso-accumbens dopamine response to stress: an electrochemical study in freely-behaving rats.

Voltammetry was used to study the role of prefrontal cortex (PFC) dopamine (DA) in modulating the nucleus accumbens (NAcc) DA response to stress. Signal increases elicited in NAcc by 15 min of restraint were monitored in freely-behaving rats following intra-PFC microinjections of D1 and D2 receptor-selective drugs. The exact site of injection was first determined by assessing the electrochemical response to stress at two dorsal-ventral levels of PFC. Consistent with previous reports, a pronounced stress response was observed ventrally at sites within the infralimbic PFC but not dorsally within the superficial layers of PFC. When microinjected into the infralimbic PFC, the D1 receptor antagonist SCH 23390 significantly enhanced the NAcc stress response. While the D1 receptor agonist SKF 38393 tended to decrease the NAcc stress response, it failed to do so reliably. Neither sulpiride (D2 receptor antagonist) nor quinpirole (D2 receptor agonist) had a significant effect. Finally, systemic administration of the selective DA uptake inhibitor GBR 12909 dose-dependently potentiated stress-induced signal increases in NAcc and in PFC, indicating that the electrochemical responses to stress in both regions were due primarily to increases in extracellular DA levels. Together, these data add to other evidence indicating that the PFC exerts an inhibitory influence on subcortical DA transmission. Specifically, the present results suggest that the NAcc DA response to stress is dampened by the concurrent activation of meso-PFC DA neurons and that this action is mediated, at least in part, by D1 receptors in PFC.

High-speed chronoamperometric measurements of mesolimbic and nigrostriatal dopamine release associated with repeated daily stress.

The effects of repeated, once daily exposure to either restraint or tail pinch stress on extracellular levels of dopamine in nucleus accumbens and striatum were electrochemically monitored in conscious rats using high-speed chronoamperometry. Acutely, both tail pinch and restraint increased extracellular dopamine levels in both regions. However, the effect of restraint on mesolimbic and, to some extent, also on nigrostriatal dopamine neurotransmission increased progressively with each daily exposure. While increases in extracellular dopamine elicited by tail pinch varied across test days, no reliable daily enhancement of electrochemical responses to this stress were observed in either of the regions studied. Pretreatment with dopamine autoreceptor-specific doses of apomorphine (50 and 100 micrograms/kg s.c.) potently inhibited stress-elicited responses in nucleus accumbens, indicating that dopamine was the primary electroactive species contributing to the electrochemical signal. The results of this study indicate that the magnitude of stress-elicited increases in levels of extracellular dopamine is determined by the number of previous exposures to stress and are consistent with reports of sensitization to the behavioral effects of stress with repeated testing. The study also provides pharmacological data that are consistent with electrophysiological evidence of increased mesolimbic dopamine cell firing during exposure to stress.

Semiquinone anion radicals formed by the reaction of quinones with glutathione or amino acids.

Using ESR spectroscopy, we show that benzoquinone, 1,4-naphthoquinone and 5-hydroxy-1,4-naphthoquinone react readily with thiol containing compounds, such as glutathione, to form their respective semiquinone anion radicals. These quinones react similarly, but less readily, with the amino group of amino acids. The therapeutic or toxicological significance of the formation of semiquinone anion radicals from the reaction of quinones with nucleophiles, such as thiols and amines, remains to be assessed.

Metabolism of 1-naphthol by tyrosinase.

1-Naphthol was metabolized by the polyphenol oxidase, tyrosinase, primarily to 1,2-naphthoquinone and to small amounts of 1,4-naphthoquinone as well as to covalently bound products. The inhibition of covalent binding by ethylenediamine, which reacts specifically with 1,2-naphthoquinone but not 1,4-naphthoquinone, suggested that most of the covalent binding was due to 1,2-naphthoquinone or a metabolite of similar structure. The activation by tyrosinase of 1-naphthol to covalently bound products suggested that it may alter the reaction kinetics of the enzyme. This was investigated by studying the effects of 1-naphthol on the tyrosinase-catalysed oxidation of 4-hydroxyanisole. Preincubation of tyrosinase with 1-naphthol increased the lag period of the oxidation of 4-hydroxyanisole, which may be due to a decrease in the amount of active enzyme, as well as to a reaction of 1-naphthol with 3,4-anisylquinone, an oxidation product of 4-hydroxyanisole. The metabolic activation of 1-naphthol by tyrosinase to covalently bound species suggests that 1-naphthol or a structurally related derivative may be of potential therapeutic application in the treatment of cells high in tyrosinase activity, such as certain melanomas.

Metabolic activation of 1-naphthol by rat liver microsomes to 1,4-naphthoquinone and covalent binding species.

1-Naphthol was metabolized by rat liver microsomes, in the presence of an NADPH-generating system, both to methanol-soluble metabolites including 1,4-naphthoquinone and an uncharacterized product(s) (X) and also to covalently bound products. NADH was much less effective as an electron donor than NADPH. Metyrapone, SKF 525-A and carbon monoxide all inhibited the metabolism of 1-naphthol to 1,4-naphthoquinone and to covalently bound products suggesting the involvement of cytochrome P-450 in at least one step in the metabolic activation of 1-naphthol to reactive products. Ethylene diamine, which reacts selectively with 1,2-naphthoquinone but not 1,4-naphthoquinone, did not affect the covalent binding whereas glutathione, which reacts with both naphthoquinones, caused an almost total inhibition of covalent binding. These and other results suggested that 1,4-naphthoquinone, or a metabolite derived from it, was responsible for most of the covalent binding observed and that little if any of the binding was due to 1,2-naphthoquinone.

Improved performance from modifications to the multidetector SPECT brain scanner.

A multidetector single photon emission computerized tomographic brain scanner was modified to improve the angular sampling. The detector plate was rotated such that 12, 24, or 36 angular projections could be acquired. Phantom experiments demonstrated that the angular aliasing artifacts seen in images obtained with 12 detectors were eliminated with 36 effective detectors. In addition, the reconstructed image noise in a uniform source was decreased by a factor of 1.7 by the use of 36 instead of 12 angular projections, as predicted by computer simulation.

Electrophoresis of reacting systems involving sulfhydryl oxidation of proteins.

Numerical solution of the relevant continuity equations has been used to examine the possible effects of intramolecular sulfhydryl oxidation on the electrophoresis of proteins. Simulations of moving boundary electrophoresis, based on variants of a previous model [J.R. Cann, N.H. Fink, and D.J. Winzor (1983) Arch. Biochem. Biophys. 221, 57-63], show that the Schlieren patterns for the ascending and descending limbs are likely to exhibit pronounced nonenantiography. Whereas the pattern for one limb may comprise essentially a single peak, that for the conjugate side can exhibit bimodality, the nature of which is time dependent. Bimodality of the Schlieren pattern can develop in either the ascending or descending limb of the electrophoresis cell, depending basically upon the number of sulfhydryl groups available for oxidation, and on the relative magnitudes of the rate constants describing the oxidation and the isomerization of the oxidized protein species. Whether the faster-moving or slower-moving peak grows with time is shown to depend upon the magnitude of the electrophoretic mobility of the resultant isomer in relation to that of the oxidized protein species. Schlieren patterns for fish muscle creatine kinase and rabbit muscle aldolase are then used to support the relevance of these predictions to moving boundary electrophoresis of proteins undergoing intramolecular sulfhydryl oxidation. Finally, numerical simulation of the zonal electrophoretic behavior of such systems serves to illustrate that bimodal patterns may also obtain, thereby giving a false impression of inherent protein heterogeneity. Emphasis is therefore placed on the importance of maintaining an adequate concentration of reducing agent throughout the medium in which the protein migrates, a potential problem in polyacrylamide gel electrophoresis at neutral pH.

The formation of active oxygen species following activation of 1-naphthol, 1,2- and 1,4-naphthoquinone by rat liver microsomes.

The hepatic microsomal metabolism of 1-naphthol, 1,2- and 1,4-naphthoquinone has been shown to generate active oxygen species by using electron spin resonance spin-trapping techniques. 1-Naphthol, in the presence of NADPH, and 1,2- and 1,4-naphthoquinone, with either NADH or NADPH, caused a stimulation in both the rate of microsomal oxygen consumption and the formation of superoxide spin adduct, 5,5-dimethyl-2-hydroxyperoxypyrrolidino-1-oxyl (DMPO-OOH). Superoxide dismutase, but not catalase, prevented the formation of this spin adduct, further supporting the suggestion that the superoxide free radical was the major oxy-radical formed during the microsomal metabolism of 1-naphthol and the naphthoquinones. These results are compatible with the suggestion that 1-naphthol may exert its toxicity to isolated hepatocytes and other cellular systems by metabolism to naphthoquinones followed by their redox cycling with concomittant generation of active oxygen species in particular superoxide free radicals.

Mechanisms of toxic injury to isolated hepatocytes by 1-naphthol.

The mechanism(s) of toxicity of 1-naphthol and two of its possible metabolites, 1,2- and 1,4-naphthoquinone, to freshly isolated rat hepatocytes has been studied. 1-Naphthol and both naphthoquinones exhibited a dose-dependent toxicity to hepatocytes. [1-14C]-1-Naphthol was metabolised by hepatocytes predominantly to its glucuronic acid and sulphate ester conjugates, but small amounts of covalently bound products were also formed. Blebbing on the surface of the hepatocytes was observed following exposure to 1-naphthol and the naphthoquinones, together with a dose-dependent decrease in intracellular glutathione (GSH), which preceded the onset of cytotoxicity. The toxicity of 1-naphthol and the naphthoquinones was potentiated by dicoumarol, an inhibitor of DT-diaphorase (NAD(P)H:quinone oxidoreductase). This enhanced toxicity was accompanied by a greater amount of surface blebbing, an increased depletion of intracellular GSH, particularly in the case of 1-naphthol and 1,4-naphthoquinone, and a decreased metabolism of 1-naphthol to its conjugates with variable effects on the amount of covalently bound products formed. These results support the suggestion that the toxicity of 1-naphthol may be mediated by the formation of 1,2-naphthoquinone and/or 1,4-naphthoquinone, which may then be metabolised by one electron reduction to naphthosemiquinone radicals. These, in turn, may covalently bind to important cellular macromolecules or enter a redox cycle with molecular oxygen thereby generating active oxygen species. Both of these processes appear to play a role in producing the cytotoxic effects of 1-naphthol.