Q: Your laboratory has pioneered new optical imaging techniques that enable you to visualize synaptic connections directly in living animals, essentially eavesdropping on the brain at work, in real time. What has surprised you most from the results you’ve seen so far?
Lichtman: Our aim in looking at nerve cells in their natural setting is precisely to learn things that could not be anticipated (and hence are surprising). We have been surprised by almost everything we have seen. Perhaps most inspiring is the sheer beauty of the cellular organization of the nervous system.
Q: How do these imaging techniques work? What key advances have led to their development?
A: The field of in vivo imaging depends on fluorescent probes that give off light of one color (say yellow) when illuminated with a different color light (say blue). The power of this approach is that by using filters that only pass the fluorescent color (e.g., yellow) a researcher can see the labeled cells on a dark background. This high contrast allows very small or dimly labeled objects to be visualized—much the same way stars are easier to see at night than during the day. Over the past several years this approach has gotten a big boost from the discovery that a gene from a bioluminescent jellyfish can be incorporated into the DNA of many cells to cause neurons in many different species (including mammals) to fluoresce. This green fluorescent protein gene has since been modified to shift the fluorescent spectrum, so that now there are also yellow, cyan, red and other color fluorescent proteins. We are now involved in generating transgenic mice that express a wide range of different fluorescent colors in individual neurons. These mice permit us to see synaptic circuitry in vivo in living animals that are anesthetized.
Q: What impact are these methods having on basic neuroscience research?
A: One critical aspect of the nervous system is its dynamic properties. For example, questions such as how long a synaptic connection lasts, or how the nervous system ages, have been difficult to study without tools that allow the same neural structures to be imaged over time. These labeling and imaging methods provide the first straightforward means of asking these questions. Although we have been doing this type of in vivo time-lapse work for over twenty years, things got much more interesting (and easier) when jellyfish genes became available.
Q: You’ve examined the process of synaptic development in laboratory animals, tracking the wiring up of the nervous system in mice over minutes, weeks and months. What has this work revealed about the fine-tuning of synapses during development?
A: We see that the final pattern of connections emerges after the vast majority of neuronal connections are trimmed away. This trimming appears to be the consequence of competition between different neurons vying to remain connected to the same target cells. Although we have studied this phenomenon in only a few parts of the nervous system, there are hints that this trimming back may be a general feature of the young brain. As this trimming occurs in the early postnatal life of a mammal, the resulting reorganization of neural circuits may allow an animal to select the subset of connections that are best tuned to the tasks the animal is being called upon to do. In this way the loss of connections may be central to learning. In the broadest sense, this idea argues that, of the many things we could become, we become who we are through a process of selective trimming back of neural circuits to a much smaller subset of brain circuits that allow us to do a few things well.
Q: You and others have begun applying these imaging methods to neurological disorders, including spinal cord injury and amyotrophic lateral sclerosis (ALS). How have they helped advance our understanding of these conditions, and do you foresee wider application to diseases?
A: Yes, the idea that diseases might be studied from the inside out by documenting the pathological processes and observing disease progression over time inside an animal allows a more direct and clearer picture of what is actually going wrong. I can imagine that virtually all disease models in animals will one day be studied this way.
For example, we’ve applied in vivo, time-lapse imaging to a mouse model of ALS to investigate how motor nerve fibers degenerate in the disease and how some of these motor axons attempt to compensate for the loss of nerve signaling from the brain. With the high-resolution images, we were able to show that there are two distinct populations of motor neurons, one group with axon branches that are clearly degenerating, and one that is undergoing compensatory reinnervation. This work suggests that if we can identify the factors that protect certain motor neurons from degenerating and/or induce their regeneration, it might be possible to design therapeutic strategies that enhance these processes.
We’ve also used these imaging methods to track the degeneration and regeneration of axons in living mice over the course of several days following spinal cord injury. We found that within 30 minutes of the injury, axons die back hundreds of micrometers, from both ends of the severed axons. In addition, many axons attempt regeneration within 6 to 24 hours of the spinal cord lesion, a growth response that begins robustly but ultimately fails, seemingly as a result of the axons’ inability to navigate in the right direction. These results show that this type of time-lapse imaging can be useful for understanding the degenerative processes that follow spinal cord injury and for evaluating therapies aimed at enhancing regeneration.