Since the deployment of the microscope by Robert Hooke in the C17th, light has been used to study cells. Most laboratories are satisfied with such passive cellular observation, however we use light not only to observe but also to stimulate cellular chemistry. This area of biophotonics is called “caged compounds”, as synthetic organic chemistry is used to make biologically signaling molecules functionally inert; irradiation liberates the caged molecules, thus “switching on” a chosen signaling pathway.
We have developed many caged compounds for stimulating both intracellular and extracellular receptors. These molecules include caged calcium, IP3, glutamate, GABA, and D-apartate. Recent work at Sinai has focused on two areas: 2-color uncaging and developing a new caging chromophore with an exceptionally large 2-photon cross section.
In 1993 the late Larry Katz published the first example of glutamate uncaging in living brain slices. I met Larry that year at CSHL where we talked about the idea of being able to perform uncaging of another biomolecule at an second wavelength. GABA was the obvious one. I became very excited by this idea but soon realised that there was no caging chromophore available that would allow this to be accomplished. Recently we have (finally) developed a new chromophore (DEAC450) that allows chromatically independent uncaging with two wavelengths.
We have used DEAC450 to cage glutamate, GABA and cAMP. Importantly five labs have tested these probes (ourselves, John Williams, Jim Surmeier, Bernardo Sabatini and Mike Higley) and confirmed the excellent properties of the new caging chromophore. Our current research focus is in this area in terms of using these probes to study dendritic integration and to develop new caged compounds.
A new caged calcium: BIST-2EGTA
Calcium is the most important signaling molecule inside cells. Fluctuations in its concentration control gene transcription, insulin secretion, muscle contraction, neurotransmission, wound healing, etc. I and my laboratory have developed four useful calcium cages: DM-nitrophen; NP-EGTA; DMNPE-4 and NDBF-EGTA. Two of my calcium cages (DM-nitrophen and NP-EGTA) have been commercially available for some time now. So, literally hundreds of biological studies have been published by us and many other groups using these probes. In particular, Erwin Neher and Bob Zucker and their former co-workers (e.g. Jens Rettig and Deiter Bruns) have used our cages to study secretion in many cells (they have published more than 60 caged calcium papers). One "problem" with these optical probes is that they are only modestly sensitive to 2-photon excitation.
In 2016 we published the design and development of a new caged Ca probe using an extended pi-electron system, that is highly sensitive to 2-photon excitation, having an absorption cross section of about 250 GM.
Importantly, BIST can be uncaging very efficiently by visible light (blue spectrum). Ca is released in less than 1 millisecond and 2-photon uncaging at 810nm is about 100x better than DM-nitrophen.
In 2019 we published the application of BIST to neurotransmitter uncaging. A very curious property of BIST-caged GABA is that adding the dendrimer cloak removed to 1P sensitivity to uncaging in aqueous solution, making it a 2P-only probe.
Uncaging in living mice
In the past 30 years the cell preparation used by neuroscientists to study neurons and glia has inevitably increased in its complexity. Initially, much important information was garnered from cultured cells studied as monolayers on glass cover slips. But this preparation does not preserve the complexity of the mammalian brain, so Bert Sakmann's lab developed the acute brain slice that has become standard in hundreds of labs around the world. In this context, we published the first unambiguous example of 2-photon uncaging in living mice in ACS Chemical Neuroscience in 2010.
Glutamate is the major transmitter in the CNS, being responsible for about 80% of transmission. In 1998 Ernst Niggli had shown that DM-nitrophen as a 2-photon caged Ca probe worked in cardiac myocytes. On the basis of these studies I made DMCNB-Glu (C below). Laser flash photolysis revealed that uncaging (=glutamate release) is very rapid (D).
In collaboration with Prof. Haruo Kasai's group, we found that 2P photolysis of DMCNB-Glu evoked rapid currents from AMPA-receptors (A). I presented these data, together with selective calcium uncaging from DMNPE-4, at the SGP conference in the Summer of 1999 (J. Gen. Physiol. 1999, 114, 1a). With this knowledge, in 2000 I synthesized MNI-glutamate, as I thought it would be useful for 2-photon photolysis (Society for Neuroscience Annual Meeting 2000, abstract 426.12-the Mill Hill group, lead by John Corrie, independently made the same compound that year). Prof. Haruo Kasai's group were the first to use MNI-glutamate for diffraction-limited, 2-photon photolysis in brain slices (Nature Neuroscience 2001). In 2007 I made a new caged glutamate (CDNI-Glu) that is 5-6 times better than MNI-glu. Before leaving Kasai's lab to start his own group in Okazaki, Masanori Matsuzaki published two elegant applications of CDNI-Glu. In 2016 Masanori moved back to Tokyo!