Capturing Thought, in Real Time

Wouldn't it be nice if we mapped how the thought processes traveled across our brain, in real time? That's exactly what Mazahir Hasan et al of Max Planck Institute for Medical Research in Heidelberg, have enabled us to view, when an action potential (AP) is underway in the central nervous system (CNS). The researchers introduced fluorescent calcium indicator proteins (FCIP) into the brain cells of mice by means of viral gene vectors. Each time an AP was underway, a lot of ionic phenomena happened. For example, the fast Sodium channels (Na+) opened (letting positive charges to the interior of the cell) leading to depolarization, Potassium (K+) channels opened (to bring back the resting membrane potential to normal, since K+ egress out of the cells) and so on.

Next , the impulse is transmitted to the post-synaptic neuron through the agency of neurotransmitters. But, for this 'coupling' between the presynaptic and postsynaptic neurons to occur; Calcium ion (Ca++) levels in the synaptic knobs of the presynaptic neurons must rise for effective degranulation of the presynaptic vesicles. And that's precisely these researchers were banking upon.

Just before the degranulation of synaptic vesicles begins; calcium ion concentration surges. Such short calcium currents peak within milliseconds, making them the appropriate ions for studying fast neuronal activity. Previously scientists had measured such currents by using microelectrodes implanted within the brain; but this method was quite unsuitable in studying moving animals or for a longer time period. So, they went on to produce stable transgenic mouse lines responding to functional calcium indicators; (including 'inverse pericam' and 'camgaroo-2') using viral vectors. These transgenic mouse lines were under TET inducible promoter (tetracycline, a broad-spectrum antibiotic) control. The TET system offered the advantage of targeting combination of different neuronal cell assemblies. The other side of the Ptetbi (bidirectional promoter tetracycline) promoter was attached to the firefly luciferase gene. They were also sensitive to doxicline (another antibiotic belonging to the same category as tetracycline) in terms of regulation of luciferase, as well.

They then used a heteromeric sensor protein called D3cpv, which was made to produce in the nerve cells of the transgenic mice. Two subunits of this protein reacted to the binding of calcium ions in a way that when the yellow-fluorescent protein (YFP) lit up and the cyan-fluorescent protein (CFP) intensity diminished. When calcium was bound to the D3cpv complex; CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) came closer together bringing about FRET, in such a way that there was a visible color change, 'visually' or optically indicating the progression of action potential in real time. CFP and YFP are spectral variants of GFP linked together by a Ca++ sensitive linker.

They used 'two-photon imaging microscopy' to study this phenomenon. They excited thinned out rat skulls using two-photons simultaneously using 'mode-locked' Titanium-sapphire laser. They then amplified the signal using photomultipliers and analyzed them.

The resolution of the experiment was limited to less than 1 Hz (frequency of action potentials). They conferred that human thought processes might be mapped in much the same 'opto-physiologic way', in contrast to the usual electrophysiologic approach. Not only does the experiment throw light on the thought processes in real-time, but also, it is expected that it will be useful in the pathophysiology and treatment of Alzheimer's disease, Parkinson's disease and Huntington's chorea.

FCIP-positive cells were found in the hippocampal CA1 and CA3 regions, mossy fiber areas of the dentate gyrus, neocortical pyramidal cells and olfactory receptor neurons, they remarked. They studied cortical pyramidal cell, olfactory and optical responses in the mice in their experiment.

Hasan, M., Friedrich, R., Euler, T., Larkum, M., Giese, G., Both, M., Duebel, J., Waters, J., Bujard, H., Griesbeck, O., Tsien, R., Nagai, T., Miyawaki, A., & Denk, W. (2004). Functional Fluorescent Ca2+ Indicator Proteins in Transgenic Mice under TET Control PLoS Biology, 2 (6) DOI: 10.1371/journal.pbio.0020163
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Reference: Damian J Wallace, Stephan Meyer zum Alten Borgloh, Simone Astori, Ying Yang, Melanie Bausen, Sebastian Kügler, Amy E Palmer, Roger Y Tsien, Rolf Sprengel, Jason N D Kerr, Winfried Denk & Mazahir T Hasan. doi:10.1038/nmeth.1242

Visualizing Viral Kinetics Using Fluorescence and Bioluminescence

It would be nice if we could see an individual virus particle, a virion, in real time within a mammalian tissue starting from its attachment to the host cell and entry, to its assembly and budding and release. The dynamics of viral production has been studied using computational models by noting the response of the virus to exogenous administration of reverse transcriptase and protease inhibitors. It was noted that a mind boggling 10^10 to 10^11 virions are produced each day by using this mathematical model. Now, Jouvenet et al have been able to fluorescently label a molecule called Gag protein (for group specific antigen), the major structural component of HIV. With the aid of fluorescence resonance energy transfer (FRET) and other techniques on these fluorescently tagged virions in living cells, they have been able to see the biogenesis of HIV virions in real time; from viral assembly to release by budding. The assembly rate accelerated as the Gag protein accumulated inside the cells. Typically, the time required for the assembly was just 5-6 minutes.

In fluorescence resonance energy transfer (FRET), an external light source shines on a donor fluorescent molecule. The donor molecule gets excited and emits light of a different frequency (fluorescence), which activate the acceptor molecule. The acceptor fluorophore then emits a photon of yet another wavelength (or a quantum, as we are referring to the particle nature of light here). Both donor and acceptor fluorophores are nothing but color variants of green fluorescent protein or GFP. The whole process (FRET) is noisy as the incident light messes up with the emitted light. The incident light may also activate the acceptor fluorophore directly, leading to error.

Recently, Asokan et al used bioluminescence from Gaussia luciferase to study adeno associated virus (AAV) kinetics in living mammalian cells. By using bioluminescent molecules, the external light source as used in FRET was no longer needed. This way, direct activation of acceptor molecule was avoided and background noise was kept to a minimum. They first amplified gLuc (Gaussia luciferase) in a plasmid by polymerase chain reaction or PCR, using primer sequences. They then fused the resulting protein to that of an adenoviral subunit of AAV, called Vp2. The resulting gLuc/AAV construct was then injected into the left hind limb of rats. They could clearly notice the AAV vector dynamics. The importance of such dynamics is realized when the use of AAV as a vector in gene therapy is considered. They opined that such a technique would be ideal in studying viral dynamics in peripheral tissues such as the eye and the brain.

Bioluminescence is used to study virus tropism and viral kinetics. Tropism refers to the different populations of host cells a virus can attack. Retroviruses have a narrow tropism meaning they can infect only a few types of cells such as CD4+ T cells and macrophages. Previous studies employed Gaussia luciferase reporter gene as a tool for studying viral dynamics. Recent experiments promise a better future for the study of viral behavior.

References:
A Asokan, J S Johnson, C Li, R J Samulski (2008). Bioluminescent virion shells: new tools for quantitation of AAV vector dynamics in cells and live animals Gene Therapy, 15 (24), 1618-1622 DOI: 10.1038/gt.2008.127

Human Immunodeficiency Virus Disease: AIDS and Related Disorders: Anthony S. Fauci, H. Clifford Lane, Harrison’s Principles of Internal Medicine, 17th Ed.

Imaging the biogenesis of individual HIV-1 virions in live cells
Nolwenn Jouvenet, Paul D. Bieniasz, & Sanford M. Simon
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