Instrument development for the analysis of low abundance analytes in single cells and small volume samples

Understanding the cell-to-cell differences between cells is important for both fundamental biology and in identifying normal and pathological functioning. Biogenic amines, which include catecholamines and indolamines, are of particular interest due to their presence throughout the central and peripheral nervous systems in many species, as well as their association with a wide variety of higher order behaviors such as sleep, memory formation, feeding, and mood; however, they are low abundance analytes since they are present in localized regions of the nervous system in femtomole to attomole quantities. Also, when sampling from the nervous system, the amines are often present within a complex matrix of proteins, salts, lipids, and other common biological compounds, which can complicate the detection and identification of trace levels of amines. This combination prompts the use of technologies that enable single cell measurements. Single cell measurements also provide insight into cell-specific metabolism, as different cell types are both quantitatively and qualitatively unique. Cell-specific metabolism distinguishes a cell that is morphologically similar to its neighbors but has a different molecular complement, which may result in a different function or indicate a difference in cell status. Differences in metabolism could also indicate potentially pathological behavior. The goal has been the design, construction, and validation of analytical instruments to enable single cell characterization. The high sensitivity and low sample consumption of capillary electrophoresis (CE) combined with the selectivity and sensitivity of laser-induced native fluorescence detection (LINF) makes CE-LINF well suited to study single cells and even subcellular organelles; however, the isolation and loading of such small samples into the CE system is challenging. This issue is addressed by designing, constructing, and interfacing a single beam optical trap with a laboratory-built CE system that uses multi-channel LINF detection, which has been optimized for single cell analyses. The optical trap is formed by tightly focusing the output of a Nd:YAG laser with a high numerical aperture objective. Once the cell is localized within the trap, the capillary inlet is moved adjacent to the trapped cell using a combination of a computer-controlled micromanipulator and a microscope stage. The cell is then released from the trap and pressure injected into the capillary. Cell lysis occurs within the capillary and the cellular constituents are subsequently separated and detected. Detection takes place using multi-channel LINF, which has been optimized for selective excitation and detection of biogenic amines. Briefly, a 224 nm HeAg hollow cathode ion laser is used in combination with a sheath-flow cuvette; the fluorescence emission is collected and measured using three channel detection with each photomultiplier tube having its own wavelength range selected with the appropriate dichroic mirror. This instrument allows unambiguous identification of a variety of catecholamines and indolamines based on differences in both their fluorescence emission profiles and migration times. This system, both as a hyphenated instrument and as individual components, has been used for several neurochemical applications, including detecting trace levels of indolamines in microdialysis samples and in single pinealocytes, the indolamine-containing cells of the pineal gland. These analyses highlight the ability of the system to isolate and manipulate single cells and perform injections and separate and detect low abundance analytes in samples with high concentrations of salts

Resonance Raman and UV-Vis Spectroscopic Characterization of FADH• in the Complex of Photolyase with UV-Damaged DNA

Escherichia coli photolyase uses blue light to repair cyclobutane pyrimidine dimers which are formed upon irradiation of DNA with ultraviolet (UV) light. E. coli photolyase is a flavoenzyme which contains a flavin adenine dinucleotide (FAD) in its active site and a 5,10-methenyltetrahydrofolate (MTHF) as a light-harvesting pigment. In the isolated enzyme, the FAD cofactor is present as a stable neutral radical semiquinone (FADH•). In this paper, we investigate the interaction between photolyase and UV-damage DNA by using resonance Raman and UV-vis spectroscopy. Substrate binding results in intensity changes and frequency shifts of the FADH• vibrations and also induces electrochromic shifts of the FADH• electronic transitions because of the substrate electric dipole moment. The intensity changes in the resonance Raman spectra can be largely explained by changes in the Raman excitation profiles because of the electrochromic shift. The size of the electrochromic shift suggests that the substrate binding geometry is similar to that of oxidized FAD in reconstituted photolyase. The frequency changes are partially a manifestation of the vibrational Stark effect induced by the substrate electric dipole moment but also because of small perturbations of the hydrogen-bonding environment of FADH• upon substrate binding. Furthermore, differences in the resonance Raman spectra of MTHF-containing photolyase and of an MTHF-less mutant suggests that MTHF may play a structural role in stabilizing the active site of photolyase while comparison to other flavoproteins indicates that the FAD cofactor has a strong hydrogen-bonding protein environment. Finally, we show that the electrochromic shift can be used as a direct method to measure photolyase-substrate binding kinetics

Resonance Raman Spectroscopic Investigation of the Light-Harvesting Chromophore in Escherichia Coli Photolyase and Vibrio Cholerae Cryptochrome-1

Photolyases and cryptochromes are flavoproteins that belong to the class of blue-light photoreceptors. They usually bind two chromophores: flavin adenine dinucleotide (FAD), which forms the active site, and a light-harvesting pigment, which is a 5,10-methenyltetrahydrofolate polyglutamate (MTHF) in most cases. In Escherichia coli photolyase (EcPhr), the MTHF cofactor is present in substoichiometric amounts after purification, while in Vibrio cholerae cryptochrome-1 (VcCry1) the MTHF cofactor is bound more strongly and is present at stoichiometric levels after purification. In this paper, we have used resonance Raman spectroscopy to monitor the effect of loss of MTHF on the protein-FAD interactions in EcPhr and to probe the protein-MTHF interactions in both EcPhr and VcCry1. We find that removal of MTHF does not perturb protein-FAD interactions, suggesting that it may not affect the physicochemical properties of FAD in EcPhr. Our data demonstrate that the pteridine ring of MTHF in EcPhr has different interactions with the protein matrix than that of MTHF in VcCry1. Comparison to solution resonance Raman spectra of MTHF suggests that the carbonyl of its pteridine ring in EcPhr experiences stronger hydrogen bonding and a more polar environment than in VcCry1, but that hydrogen bonding to the pteridine ring amine hydrogens is stronger in VcCry-1. These differences in hydrogen bonding may account for the higher binding affinity of MTHF in VcCry1 compared to EcPhr

Signals from the Brainstem Sleep/Wake Centers Regulate Behavioral Timing via the Circadian Clock

<div><p>Sleep-wake cycling is controlled by the complex interplay between two brain systems, one which controls vigilance state, regulating the transition between sleep and wake, and the other circadian, which communicates time-of-day. Together, they align sleep appropriately with energetic need and the day-night cycle. Neural circuits connect brain stem sites that regulate vigilance state with the suprachiasmatic nucleus (SCN), the master circadian clock, but the function of these connections has been unknown. Coupling discrete stimulation of pontine nuclei controlling vigilance state with analytical chemical measurements of intra-SCN microdialysates in mouse, we found significant neurotransmitter release at the SCN and, concomitantly, resetting of behavioral circadian rhythms. Depending upon stimulus conditions and time-of-day, SCN acetylcholine and/or glutamate levels were augmented and generated shifts of behavioral rhythms. These results establish modes of neurochemical communication from brain regions controlling vigilance state to the central circadian clock, with behavioral consequences. They suggest a basis for dynamic integration across brain systems that regulate vigilance states, and a potential vulnerability to altered communication in sleep disorders.</p></div

LDTg stimulation resets phasing of behavioral rhythms during subjective night.

<p>LDTg stimulation (150 µA, 10 Hz, 2-msec pulse duration, 20 min) at CT 14 and 15 (<b>D</b>) delays circadian behavioral rhythms while stimulation at CT 0 (<b>H</b>) advances circadian rhythms. <b>B</b>, <b>D</b>, <b>F</b>, and <b>H</b> are representative actograms following LDTg stimulations, while <b>A, C, E</b>, and <b>G</b> are representative actograms following sham stimulations. ∇ indicates treatment time. Gray lines indicate onset of activity before and after treatment. <b>I</b> depicts the average of all treatments. Numbers above or below each bar indicate number of animals in each treatment. * indicates p<0.05 by Two-Way ANOVA with Holm-Sidak <i>post hoc</i> test.</p

LDTg or PPTg stimulation increases glutamate or ACh at the SCN in early night.

<p>Following LDTg (<b>A</b>) or PPTg (<b>B</b>) stimulation with Condition 1 (150 µA, 10 Hz, 2-msec pulse duration), ACh significantly increases.PPTg stimulation with Condition 2 (<b>C</b>, 40 µA, 0.2 Hz, 0.2 msec pulse duration) significantly increases glutamate (Glu) release at the SCN. PPTg stimulation with Condition 3 (<b>D</b>, 400 µA, 60 Hz, 0.2 msec pulse duration, applied as a 1-sec train every min) significantly increases ACh release at the SCN. * indicates p<0.05 by Two-Way ANOVA with Holm-Sidak <i>post hoc</i> test.</p

Early evening PPTg stimulus conditions favoring glutamate <i>vs.</i> ACh release at SCN alters behavioral rhythms.

<p>At CT 15, stimuli that increase glutamate (Glu) release (0.2 Hz, 40 µA, 0.2-msec pulse duration) or Glu and ACh release (10 Hz, 150 µA, 2-msec pulse duration) delay circadian behavioral rhythms. However, stimulus conditions that raise only ACh levels (60 Hz, 400 µA, 0.2-msec pulse duration) do not alter phasing of circadian rhythms at any time tested. <b>A, C, E</b>, and <b>G</b> are representative actograms from animals receiving 0.2-Hz stimulations. <b>B, D, F</b>, and <b>H</b> are representative actograms from animals receiving 60-Hz stimulations. ∇ indicates treatment time. Gray lines indicate onset of activity before and after treatments. <b>I</b> depicts the average of all treatments. Number of animals in each treatment appears above or below the bar. * indicates p<0.05 by Two-Way ANOVA with Holm-Sidak <i>post-hoc</i> test.</p

Electrical stimulation of LDTg or PPTg increases ACh at the SCN.

<p>Stimulating LDTg (<b>A</b>) or PPTg (<b>B</b>) at 150 µA, 10 Hz, 2-msec pulse duration causes significant increase in ACh levels in SCN dialysate. When LDTg (<b>C</b>) or PPTg (<b>D</b>) is stimulated at different times of day, SCN levels of ACh increased significantly at all times tested. The magnitude of response across different times of day is not significant. Black bars denote times of stimulation; significance level is marked (* = p<0.05, ** = p<0.001, Two-Way ANOVA with Holm-Sidak <i>post hoc</i> test). ACh, acetylcholine; LDTg, laterodorsal tegmental nucleus; PPTg, pedunculopontine tegmental nucleus; SCN, suprachiasmatic nucleus; ZT, Zeitgeber Time.</p