Greater ethanol inhibition of presynaptic dopamine release in C57BL/6J than DBA/2J mice: Role of nicotinic acetylcholine receptors.

The mesolimbic dopamine system, originating in the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc), has been heavily implicated in the reinforcing effects of ethanol. Recent slice voltammetry studies have shown that ethanol inhibits dopamine release selectively during high-frequency activity that elicits phasic dopamine release shown to be important for learning and reinforcement. Presently, we examined ethanol inhibition of electrically evoked NAc dopamine in two mouse strains with divergent dopamine responses to ethanol, C57BL/6 (C57) and DBA/2J (DBA) mice. Previous electrophysiology and microdialysis studies have demonstrated greater ethanol-induced VTA dopaminergic firing and NAc dopamine elevations in DBA compared to C57 mice. Additionally, DBA mice have greater ethanol responses in dopamine-related behaviors, including hyperlocomotion and conditioned place preference. Currently, we demonstrate greater sensitivity of ethanol inhibition of NAc dopamine signaling in C57 compared to DBA mice. The reduced sensitivity to ethanol inhibition in DBA mice may contribute to the overall greater ethanol-induced dopamine signaling and related behaviors observed in this strain. NAc cholinergic activity is known to potently modulate terminal dopamine release. Additionally, ethanol is known to interact with multiple aspects of nicotinic acetylcholine receptor activity. Therefore, we examined ethanol-mediated inhibition of dopamine release at two ethanol concentrations (80 and 160 mM) during bath application of the non-selective nicotinic receptor antagonist mecamylamine, as well as compounds selective for the ß2-(dihydro-ß-erythroidine hydrobromide; DhßE) and α6-(α-conotoxin MII [H9A; L15A]) subunit-containing receptors. Mecamylamine and DhßE decreased dopamine release and reduced ethanol's inhibitory effects on dopamine in both DBA and C57 mice. Further, α-conotoxin also reduced the dopamine release and the dopamine-inhibiting effects of ethanol at the 80 mM, but not 160 mM, concentration. These data suggest that ethanol is acting in part through nicotinic acetylcholine receptors, or downstream effectors, to reduce dopamine release during high-frequency activity.

The human immunodeficiency virus-1-associated protein, Tat1-86, impairs dopamine transporters and interacts with cocaine to reduce nerve terminal function: a no-net-flux microdialysis study.

Injection drug use accounts for approximately one-third of human immunodeficiency virus (HIV) infections in the United States. HIV-associated proteins have been shown to interact with various drugs of abuse to incite concerted neurotoxicity. One common area for their interaction is the nerve terminal, including dopamine transporter (DAT) systems. However, results regarding DAT function and regulation in HIV-infection, regardless of drug use, are mixed. Thus, the present experiments were designed to explicitly control Tat and cocaine administration in an in vivo rat model in order to reconcile differences that exist in the literature to date. We examined Tat plus cocaine-induced alterations using no-net-flux microdialysis, which is sensitive to alterations in DAT function, in order to test the potential for DAT as an early mediator of HIV-induced oxidative stress and neurodegeneration in vivo. Within 5 h of intra-accumbal administration of the HIV-associated protein, Tat, we noted a significant reduction in local DAT efficiency with little change in DA overflow/release dynamics. Further, at 48 h post-Tat administration, we demonstrated a concerted effect of the HIV-protein Tat with cocaine on both uptake and release function. Finally, we discuss the extent to which DAT dysfunction may be considered a predecessor to generalized nerve terminal dysfunction. Characterization of DAT dysfunction in vivo may provide an early pharmacotherapeutic target, which in turn may prevent or attenuate downstream mediators of neurotoxicity (i.e., reactive species) to dopamine systems occurring in neuro-AIDS.

Algal species and light microenvironment in a low-pH, geothermal microbial mat community.

Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to < 1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. A Cyanidioschyzon merolae sequence was predominant at temperatures of > or = 49 degrees C. A Chlorella protothecoides var. acidicola sequence and a Paradoxia multisita-like sequence were predominant at temperatures of < or = 39 degrees C.

Measuring total soil carbon with laser-induced breakdown spectroscopy (LIBS).

Improving estimates of carbon inventories in soils is currently hindered by lack of a rapid analysis method for total soil carbon. A rapid, accurate, and precise method that could be used in the field would be a significant benefit to researchers investigating carbon cycling in soils and dynamics of soil carbon in global change processes. We tested a new analysis method for predicting total soil carbon using laser-induced breakdown spectroscopy (LIBS). We determined appropriate spectral signatures and calibrated the method using measurements from dry combustion of a Mollisol from a cultivated plot. From this calibration curve we predicted carbon concentrations in additional samples from the same soil and from an Alfisol collected in a semiarid woodland and compared these predictions with additional dry combustion measurements. Our initial tests suggest that the LIBS method rapidly and efficiently measures soil carbon with excellent detection limits (approximately 300 mg/kg), precision (4-5%), and accuracy (3-14%). Initial testing shows that LIBS measurements and dry combustion analyses are highly correlated (adjusted r2 = 0.96) for soils of distinct morphology, and that a sample can be analyzed by LIBS in less than one minute. The LIBS method is readily adaptable to a field-portable instrument, and this attribute--in combination with rapid and accurate sample analysis--suggests that this new method offers promise for improving measurement of total soil carbon. Additional testing of LIBS is required to understand the effects of soil properties such as texture, moisture content, and mineralogical composition (i.e., silicon content) on LIBS measurements.

Highly ordered vertical structure of Synechococcus populations within the one-millimeter-thick photic zone of a hot spring cyanobacterial mat.

A variety of contemporary techniques were used to investigate the vertical distribution of thermophilic unicellular cyanobacteria, Synechococcus spp., and their activity within the upper 1-mm-thick photic zone of the mat community found in an alkaline siliceous hot spring in Yellowstone National Park in Wyoming. Detailed measurements were made over a diel cycle at a 61 degrees C site. Net oxygenic photosynthesis measured with oxygen microelectrodes was highest within the uppermost 100- to 200-microm-thick layer until midmorning, but as the day progressed, the peak of net activity shifted to deeper layers, stabilizing at a depth of 300 microm from midday throughout the afternoon. Examination of vertical thin sections by bright-field and autofluorescence microscopy revealed the existence of different populations of Synechococcus which form discrete bands at different vertical positions. Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments from horizontal cryosections obtained at 100-microm-thick vertical intervals also suggested vertical stratification of cyanobacterial, green sulfur bacterium-like, and green nonsulfur bacterium-like populations. There was no evidence of diel migration. However, image analysis of vertical thin sections revealed the presence of a narrow band of rod-shaped Synechococcus cells in which the cells assumed an upright position. These upright cells, located 400 to 800 microm below the surface, were observed only in mat samples obtained around noon. In mat samples obtained at other time points, the cells were randomly oriented throughout the mat. These combined observations reveal the existence of a highly ordered structure within the very thin photic zone of this hot spring microbial mat, consisting of morphologically similar Synechococcus populations that are likely to be differentially adapted, some co-occurring with green sulfur bacterium-like populations, and all overlying green nonsulfur bacterium-like populations.

A natural view of microbial biodiversity within hot spring cyanobacterial mat communities.

This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats.

Light-induced motility of thermophilic Synechococcus isolates from Octopus Spring, Yellowstone National Park.

This study demonstrates light-induced motility of two thermophilic Synechococcus isolates that are morphologically similar but that belong to different cyanobacterial lineages. Both isolates migrated away from densely inoculated streaks to form fingerlike projections extending toward or away from the light source, depending on the light intensity. However, the two isolates seemed to prefer widely different light conditions. The behavior of each isolate was controlled by several factors, including temperature, preacclimation of inocula, acclimation during the experiment, and strain-specific genetic preferences for different light conditions (adaptation). Time-lapse microscopy confirmed that these projections were formed by actively gliding cells and were not simply the outcome of directional cell division. The observed motility rates of individual cells of 0.1 to 0.3 micrometers s-1 agreed well with the distance traversed by the projections, 0.3 to 0.5 mm h-1, suggesting that most cells in each projection are travelling in the same direction. The finding of motility among two phylogenetically unaffiliated unicellular cyanobacteria suggests that this trait may be widespread among this group. If so, this would have important implications for experiments on colonization, succession, diel positioning, and photosynthetic activity in hot spring mats dominated by Synechococcus-like cyanobacteria.

Population structure and physiological changes within a hot spring microbial mat community following disturbance.

The influence of disturbance on a hot spring cyanobacterial mat community was investigated by physically removing the top 3.0 mm, which included the entire cyanobacterial layer. Changes in 16S rRNA-defined populations were monitored by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments. Some previously absent cyanobacterial populations colonized the disturbed areas, while some populations which were present before the disturbance remained absent for up to 40 days. Changes in physiological activity were measured by oxygen microelectrode analyses and by 14CO2 incorporation into cyanobacterial molecular components. These investigations indicated substantial differences between the disturbed and undisturbed mats, including an unexplained light-induced oxygen consumption in the freshly exposed mat, increased carbon partitioning by phototrophs into growth-related macromolecules, bimodal vertical photosynthesis profiles, and delayed recovery of respiration relative to photosynthesis.

Seasonal distributions of dominant 16S rRNA-defined populations in a hot spring microbial mat examined by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments was used to examine the distributions of bacterial populations within a hot spring microbial mat (Octopus Spring, Yellowstone National Park). Populations at sites along the thermal gradient of the spring's effluent channel were surveyed at seasonal intervals. No shift in the thermal gradient was detected, and populations at spatially or temperature-defined sites exhibited only slight changes over the annual sampling period. A new cyanobacterial 16S rRNA sequence type was detected at temperatures from 63 to 75 degrees C. A new green nonsulfur bacterium-like sequence type was also detected at temperatures from 53 to 62 degrees C. Genetically unique though closely related cyanobacterial and green nonsulfur bacterium-like populations were successively distributed along the thermal gradient of the Octopus Spring effluent channel. At least two cyanobacterial populations were detected at each site; however, a limited ability to detect some cyanobacterial populations suggests that only dominant populations were observed.

Biodiversity within hot spring microbial mat communities: molecular monitoring of enrichment cultures.

We have begun to examine the basis for incongruence between hot spring microbial mat populations detected by cultivation or by 16S rRNA methods. We used denaturing gradient gel electrophoresis (DGGE) to monitor enrichments and isolates plated therefrom. At near extincting inoculum dilutions we observed Chloroflexus-like and cyanobacterial populations whose 16S rRNA sequences have been detected in the 'New Pit' Spring Chloroflexus mat and the Octopus Spring cyanobacterial mat. Cyanobacterial populations enriched from 44 to 54 degrees C and 56 to 63 degrees C samples at near habitat temperatures were similar to those previously detected in mat samples of comparable temperatures. However, a lower temperature enrichment from the higher temperature sample selected for the populations found in the lower temperature sample. Three Thermus populations detected by both DGGE and isolation exemplify even more how enrichment may bias our view of community structure. The most abundant population was adapted to the habitat temperature (50 degrees C), while populations adapted to 65 degrees C and 70 degrees C were 10(2)- and 10(4)-fold less abundant, respectively. However, enrichment at 70 degrees C favored the least abundant strain. Inoculum dilution and incubation at the habitat temperature favored the more numerically relevant populations. We enriched many other aerobic chemoorganotrophic populations at various inoculum dilutions and substrate concentrations, most of whose 16S rRNA sequences have not been detected in mats. A common feature of numerically relevant cyanobacterial, Chloroflexus-like and aerobic chemorganotrophic populations, is that they grow poorly and resist cultivation on solidified medium, suggesting plating bias, and that the medium composition and incubation conditions may not reflect the natural microenvironments these populations inhabit.

Enrichment culture and microscopy conceal diverse thermophilic Synechococcus populations in a single hot spring microbial mat habitat.

Recent molecular studies have shown a great disparity between naturally occurring and cultivated microorganisms. We investigated the basis for disparity by studying thermophilic unicellular cyanobacteria whose morphologic simplicity suggested that a single cosmopolitan species exists in hot spring microbial mats worldwide. We found that partial 16S rRNA sequences for all thermophilic Synechococcus culture collection strains from diverse habitats are identical. Through oligonucleotide probe analysis and cultivation, we provide evidence that this species is strongly selected for in laboratory culture to the exclusion of many more-predominant cyanobacterial species coexisting in the Octopus Spring mat in Yellowstone National Park. The phylogenetic diversity among Octopus Spring cyanobacteria is of similar magnitude to that exhibited by all cyanobacteria so far investigated. We obtained axenic isolates of two predominant cyanobacterial species by diluting inocula prior to enrichment. One isolate has a 16S rRNA sequence we have not yet detected by cloning. The other has a 16S rRNA sequence identical to a new cloned sequence we report herein. This is the first cultivated species whose 16S rRNA sequence has been detected in this mat system by cloning. We infer that biodiversity within this community is linked to guild structure.

Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments was used to profile microbial populations inhabiting different temperature regions in the microbial mat community of Octopus Spring, Yellowstone National Park. DGGE allowed a rapid evaluation of the distributions of amplifiable sequence types. Profiles were essentially identical within regions of the mat defined by one temperature range but varied between sites with different temperature ranges. Individual DGGE bands were sequenced, and the sequences were compared with those previously obtained from the mat by cloning and from cultivated Octopus Spring isolates. Two known cyanobacterial populations and one known green nonsulfur bacterium-like population were detected by DGGE, as were many new cyanobacterial and green nonsulfur and green sulfur bacterium-like populations and a novel bacterial population of uncertain phylogenetic affiliation. The distributions of several cyanobacterial populations compared favorably with results obtained previously by oligonucleotide probe analyses and suggest that adaptation to temperature has occurred among cyanobacteria which are phylogenetically very similar.

Method for isolation and purification of cyanobacteria.

A method employing nutrient saturated glass fiber filters allowed the isolation of the same numbers of cyanobacteria from freshwater as were obtained with medium solidified with agar, while providing a 2- to 15-fold reduction in the number of accompanying heterotrophic bacteria. Imipenem, a broad-spectrum beta-lactam antibiotic which inhibits peptidoglycan biosynthesis, was superior to some other beta-lactam antibiotics for reducing the numbers of heterotrophic bacterial contaminants associated with freshly isolated cyanobacteria to a level which facilitated the production of axenic cyanobacterial cultures.