Sanjeev Kumar Kaushalya

Protein aggregation probed by two-photon fluorescence correlation spectroscopy of native tryptophan.

Fluorescence correlation spectroscopy (FCS) has proven to be a powerful tool for the study of a range of biophysical problems including protein aggregation. However, the requirement of fluorescent labeling has been a major drawback of this approach. Here we show that the intrinsic tryptophan fluorescence, excited via a two-photon mechanism, can be effectively used to study the aggregation of tryptophan containing proteins by FCS. This method can also yield the tryptophan fluorescence lifetime in parallel, which provides a complementary parameter to understand the aggregation process. We demonstrate that the formation of soluble aggregates of barstar at pH 3.5 shows clear signatures both in the two-photon tryptophan FCS data and in the tryptophan lifetime analysis. The ability to probe the soluble aggregates of unmodified proteins is significant, given the major role played by this species in amyloid toxicity.

Three-photon microscopy shows that somatic release can be a quantitatively significant component of serotonergic neurotransmission in the mammalian brain

Recent experiments on monoaminergic neurons have shown that neurotransmission can originate from somatic release. However, little is known about the quantity of monoamine available to be released through this extrasynaptic pathway or about the intracellular dynamics that mediate such release. Using three‐photon microscopy, we directly imaged serotonin autofluorescence and investigated the total serotonin content, release competence, and release kinetics of somatic serotonergic vesicles in the dorsal raphe neurons of the rat. We found that the somata of primary cultured neurons contain a large number of serotonin‐filled vesicles arranged in a perinuclear fashion. A similar distribution is also observed in fresh tissue slice preparations obtained from the rat dorsal raphe. We estimate that the soma of a cultured neuron on an average contains about 9 fmoles of serotonin in about 450 vesicles (or vesicle clusters) of ≤370 nm average diameter. A substantial fraction (>30%) of this serotonin is released with a time scale of several minutes by K+‐induced depolarization or by para‐chloroamphetamine treatment. The amount of releasable serotonin stored in the somatic vesicles is comparable to the total serotonin content of all the synaptic vesicles in a raphe neuron, indicating that somatic release can potentially play a major role in serotonergic neurotransmission in the mammalian brain.

Quantitative measurement of serotonin synthesis and sequestration in individual live neuronal cells

Synthesis and subsequent sequestration into vesicles are essential steps that precede neurotransmitter exocytosis, but neither the total neurotransmitter content nor the fraction sequestered into vesicles have been measured in individual live neurons. We use multiphoton microscopy to directly observe intracellular and intravesicular serotonin in the serotonergic neuronal cell line RN46A. We focus on how the relationship between synthesis and sequestration changes as synthesis is up‐regulated by differentiation or down‐regulated by chemical inhibition. Temperature‐induced differentiation causes an increase of about 60% in the total serotonin content of individual cells, which goes up to about 10 fmol. However, the number of vesicles per cell increases by a factor of four and the proportion of serotonin sequestered inside the vesicles increases by a factor of five. When serotonin synthesis is inhibited in differentiated cells and the serotonin content goes down to the level present in undifferentiated cells, the sequestered proportion still remains at this high level. The total neurotransmitter content of a cell is, thus, an unreliable indicator of the sequestered amount.

Optimized Derivation and Functional Characterization of 5-HT Neurons from Human Embryonic Stem Cells

The ability to study the characteristics of serotonin release from human serotonergic neurons is valuable both in terms of understanding disease pathology and in trying to understand how drugs that affect the serotonergic system alter neurotransmitter release. There is, however, no good in vitro system to model human serotonergic neurons. Although human embryonic stem (hES) cells offer an attractive model system, the derivation of serotonergic neurons from these cells has remained at a low efficiency. To address this problem, Nestin positive precursors from HUES7 hES cell line were first generated. These Nestin positive cells when terminally differentiated gave rise to 20% MAP-2 positive neurons. A high percentage (>40%) of these neurons could be converted to serotonergic neurons. These serotonergic neurons expressed both serotonin and the neuron-specific tryptophan hydroxylase enzyme. In addition, they expressed several of the transcription factors that have been associated with serotonergic differentiation including Mash1 and Pet1. Finally, during the process of neuronal differentiation, the serotonin content, the localization of serotonin vesicles, and their ability to release serotonin following depolarization was characterized using a live cell serotonin imaging technique based on three-photon microscopy. Thus, for the first time, we demonstrate the feasibility of characterizing the development and function of human serotonergic neurons in vitro.

Microfluorometric detection of catecholamines with multiphoton-excited fluorescence

We demonstrate sensitive spatially resolved detection of physiological chromophores that emit in the ultraviolet (<330 nm). An atypical laser source (a visible wavelength femtosecond optical parametric oscillator), and an unconventional collection geometry (a lensless detector that detects the forward-emitted fluorescence) enable this detection. We report the excitation spectra of the catecholamines dopamine and norepinephrine, together with near-UV emitters serotonin and tryptophan, in the range of 550-595 nm. We estimate the molecular two-photon action cross section of dopamine, norepinephrine, and serotonin to be 1.2 mGM (1 GM, or Goppert Mayor, is equal to 10(-58) m4 s(-1) photon(-1)), 2 mGM, and 43 mGM, respectively, at 560 nm. The sensitivity achieved by this method holds promise for the microscopic imaging of vesicular catecholamines in live cells.

A high-resolution large area serotonin map of a live rat brain section.

We employ three-photon microscopy to produce a high-resolution map of serotonin autofluorescence in a rat midbrain section (covering more than half of the brain), to quantitatively characterize serotonin distribution and release in different areas of a live brain slice. The map consists of a tiling of ∼160 contiguous optical images (covering an area of ∼27 mm2 with sub-μm resolution in 20 min), and is recorded before and after inducing depolarization. We observe that the total serotonin exocytosed from the somata in the raphe is quantitatively comparable with regions containing a high density of serotonergic processes. Our results demonstrate that high-resolution, wide-area, dynamic neurotransmitter mapping is now possible.

The Dynamics of Somatic Exocytosis in Monoaminergic Neurons

Some monoaminergic neurons can release neurotransmitters by exocytosis from their cell bodies. The amount of monoamine released by somatic exocytosis can be comparable to that released by synaptic exocytosis, though neither the underlying mechanisms nor the functional significance of somatic exocytosis are well understood. A detailed examination of these characteristics may provide new routes for therapeutic intervention in mood disorders, substance addiction, and neurodegenerative diseases. The relatively large size of the cell body provides a unique opportunity to understand the mechanism of this mode of neuronal exocytosis in microscopic detail. Here we used three photon and total internal reflection fluorescence microscopy to focus on the dynamics of the pre-exocytotic events and explore the nature of somatic vesicle storage, transport, and docking at the membrane of serotonergic neurons from raphe nuclei of the rat brain. We find that the vesicles (or unresolved vesicular clusters) are quiescent (mean square displacement, MSD ∼0.04 μm2/s) before depolarization, and they move minimally (<1 μm) from their locations over a time-scale of minutes. However, within minutes of depolarization, the vesicles become more dynamic (MSD ∼0.3 μm2/s), and display larger range (several μm) motions, though without any clear directionality. Docking and subsequent exocytosis at the membrane happen at a timescale (∼25 ms) that is slower than most synaptic exocytosis processes, but faster than almost all somatic exocytosis processes observed in endocrine cells. We conclude that, (A) depolarization causes de-tethering of the neurotransmitter vesicles from their storage locations, and this constitutes a critical event in somatic exocytosis; (B) their subsequent transport kinetics can be described by a process of constrained diffusion, and (C) the pre-exocytosis kinetics at the membrane is faster than most other somatic exocytosis processes reported so far.

Label-free dopamine imaging in live rat brain slices.

Dopaminergic neurotransmission has been investigated extensively, yet direct optical probing of dopamine has not been possible in live cells. Here we image intracellular dopamine with sub-micrometer three-dimensional resolution by harnessing its intrinsic mid-ultraviolet (UV) autofluorescence. Two-photon excitation with visible light (540 nm) in conjunction with a non-epifluorescent detection scheme is used to circumvent the UV toxicity and the UV transmission problems. The method is established by imaging dopamine in a dopaminergic cell line and in control cells (glia), and is validated by mass spectrometry. We further show that individual dopamine vesicles/vesicular clusters can be imaged in cultured rat brain slices, thereby providing a direct visualization of the intracellular events preceding dopamine release induced by depolarization or amphetamine exposure. Our technique opens up a previously inaccessible mid-ultraviolet spectral regime (excitation ~270 nm, emission < 320 nm) for label-free imaging of native molecules in live tissue.

Serotonin: multiphoton imaging and relevant spectral data

Coupling three-photon microscopy with automated stage movement can now produce a live high resolution map of the neurotransmitter serotonin in a single cross section of the whole rat brain. Accurate quantification of these serotonin images demands appropriate spectral filtering. This requires one to consider that the spectral characteristics of serotonin show a remarkable variation as it non-covalently associates with different molecules, as we discuss here. Also it is known that serotonin emission changes when it forms a covalent adduct with para-formaldehyde. This provides a potential route for producing a whole brain serotonin map using multiphoton microscopy in a fixed rat brain. Here we take the initial step showing that multiphoton microscopy of this adduct can quantitatively image chemically induced changes in serotonin distribution.

Multiphoton ultraviolet microscopy images dopamine dynamics in live brain tissue

The selection of NEs based on sensitivity analysis, employing Artificial Neural Network (ANN) using NeuroSolutions V5, was conceded. Ligand selection was then followed by a detailed interaction studies using a more focused fragment-based geometrical optimization (isobolographic analysis) which was further later substantiated through the use of docking studies (Glide 4.0) and design of experiments. Results and discussion: Curcumin and glycosylated nornicotine demonstrated higher sensitivities toward energy minimizations with A P based upon the Mean Square Error and input–output mapping via ANN (Fig. 2). Interestingly, curcumin formed Hbonds with the alanine residues and were capable of binding to the aliphatic amino acids residues (A 12–28; Glide score: −3.79) whereas the glucose side-chain of glycosylated nornicotine exhibited H-bonding with histidine and phenylalanine (AMBER) and with glutamine, phenylalanine and aspartic acid during the docking studies (Glide score: −2.89; Fig. 3). A highly synergistic interaction was observed, using an isobolographic analysis and Loewe additivity relationship (Quantitative parameter ( ): 0.45),displaying a possible reduction in individual effective concentration by a factor of 4 and 5, respectively, without compromising and even substantiating the therapeutic benefit. A neuroprotective combination of three molecules of Curcumin and three molecules of Glyconornicotine was proposed by the DoE model indicating a possible 1:1 combination with maximum of three molecules of each NE per A P oligomer. Conclusion: Our work offers a novel mathematical and in silico approach that constitutes a new frontier in providing neuroscientists with a template for in vitro and in vivo molecular experimentation.