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From surrogate hemodynamics-based fMRI towards direct functional imaging of neural activity via sensing activity-induced cell swellings and neurotransmitter releases in vivo

Periodic Reporting for period 1 - SPECIFIC fMRI (From surrogate hemodynamics-based fMRI towards direct functional imaging of neural activity via sensing activity-induced cell swellings and neurotransmitter releases in vivo)

Reporting period: 2015-04-01 to 2017-03-31

The Marie Sklodowksa Curie proposal revolved around developing new methods to quantify neural activity more directly then the state-of- the-art hemodynamic-based MRI methods. We proposed to develop microstructural methods capable of resolving cell swellings upon activation; methods that would sense neurotransmitter releases using advanced Magnetic Resonance Spectroscopy; and investigating widespread networks with optogenetics and these new methods. Achieving these goals would impact society by enabling a better investigation of neural activity in-vivo – a target that insofar has never been achieved.
Our efforts in this direction were quite extensive. First, we realized that we needed much higher temporal resolution than initially thought, because the requirement of acquiring microstructural MRI (via the NOGSE sequence proposed) requires at least two images. However, we found that since those images always had to be separated by 1-2 seconds, our results were compromised. To overcome this, we developed a new method termed “INDI-MRI” – a new way of capturing fast microstructural dynamics. Indeed, the development of INDI was the “missing link” in terms of getting the proposed NOGSE sequence to work in-vivo. These efforts culminated in a recent publication in Magnetic Resonance in Medicine (Ianus and Shemesh, Incomplete Initial Nutation Diffusion Imaging: an ultrafast, single-scan approach for diffusion mapping, Magnetic Resonance in Medicine, in-press 2017).
Furthermore, we have had to understand mixing time effects from the dual waveform nature of NOGSE. To this end, we developed a new pulse sequence termed DODE – a variation on NOGSE – which helped us understand these effects. The findings were published in Ianus, Shemesh, Alexand, Drobnjak, Double Oscillating Diffusion Encoding and Sensitivity to Microscopic Anisotropy, Magnetic Resonance in Medicine 2017;78:550–564. Even after termination of the grant, we continue our endeavor to develop the NOGSE sequence based on INDI-MRI and DODE, and so this has served immensely for the purpose of the grant.
Another main effort was the acquisition of spectroscopic data during activation. In this avenue, we have performed experiments using forepaw stimulation as a “sanity-check”, prior to the application of optogenetics. We were successful in measuring Glu and GABA variations upon forepaw stimulation (paper being written). As well, since an ERC grant on a similar topic replaced the Marie Curie, we acknowledged it in a peer-reviewed conference publication (Shemesh et al, Primary neurotransmitter variations upon forepaw stimulation revealed by functional Magnetic Resonance Spectroscopy in the rat, International Society for Magnetic Resonance in Medicine 2017). Ongoing efforts in the Lab are now underway to implement the more advanced pulse sequences and mapping the Glu/GABA variations in optogenetic stimulations.
The last part of the proposal involving stroboscopic acquisitions is also ongoing work, but since the grant was terminated quite rapidly, we did not have the chance to develop the final objective fully.
Nevertheless, we have (a) successfully implemented optogenetics in the Lab, and it is nowadays being routinely used for many types of study, including of course, the Marie Curie-funded study; (b) we have implemented the setup for performing MRI in fully awake animals. This involved extensive training of animals to the magnet environment including exposure and reward presentation in the scanner such that the animals feel the MRI scanner is a safe and welcoming environment despite of the loud noises and sounds. Behavioral and chemical tests performed show that the animals are not different in any trait when compared to control subjects that have not undergone training. And so, the third objective, while not fully realized, has been dramatically worked on and developed. We will report these results in due course and of course are still committed to perform the proposed experiments.
In addition to the above-mentioned achievements of the grant’s objectives, we also side-tracked slightly to understand a few basic mechanisms related with the underlying microstructure, that were important for interpretation of the results arising from NOGSE or INDI experiments. First, we discovered that we had to understand the underlying axonal densities, as those would contribute to the axonal conductance and hence affect our results (albeit indirectly). We thus had to develop a method capable of mapping the ensities, which we have achieved and published in 2017 (Nunes, Cruz, Jespersen, Shemesh, Mapping axonal density and average diameter using non-monotonic time-dependent gradient-echo MRI, Journal of Magnetic Resonance 2017; 277: 117–130). Similarly, we had to understand the time-dependence of the diffusion properties in those axons, and an extensive study demonstrating how the diffusion properties vary with diffusion time resulted in an accepted paper (Jespersen, Lynge, Hansen, Shemesh, Diffusion time dependence of microstructural parameters in fixed spinal cord, NeuroImage, in-press 2017
The grant has been cut short, but all told, we have made extensive progress towards achieving its goals. First, the development of INDI-MRI was a major step beyond the state-of- the-art, and now facilitates the acquisition of hemodynamic and microstructural maps nearly simultaneously, a property that is now in use in our Lab for mapping axonal swellings. Indeed, preliminary results are extremely promising and we are confident that the component of the grant in terms of mapping axonal swellings is very close to being achieved. Furthermore, INDI-MRI can parallelize acquisitions of conventional diffusion MRI, which we expect to impart an impact in clinical scanners for use in hospitals and research alike. Similarly, the goal of investigating neurotransmitter release has been largely realized in the sense that we have been able to detect variations in the primary neurotransmitters using advanced spectroscopic methods; further studies are now required to elucidate the origin of the signal differences in Glutamate and GABA signals that we have measured. The impact of this finding relates with (a) confirming the ability of detecting such important signals at ultrahigh fields and (b) providing an interesting window for neuroscientists and medical doctors to investigate subjects using this approach.
It can also serve as a driver for industry – and hence also impact society – and encourage developments in high field MRI scanners, because detecting neurotransmitters with good resolution requires high field systems.
To summarize, the grant was quite a success and we are indebted to the MSC Action for its generosity. Thank you!