Figure 54 Rita Levi Montalcini in 1977 From Purves and Lichtman 1985

The Peripheral Neuropathy Solution

Peripheral Neuropathy Program By Dr. Randall Labrum

Get Instant Access

What I learned from Hamburger about neural development and nerve growth factor during the next few years had a significant impact on what was going on in my lab, where people were toiling away on the formation and maintenance of synaptic connections in the simple and accessible systems that various autonomic ganglia in mammals provided. By then, Levi-Montalcini was spending most of her time in Rome, despite her appointment in St. Louis, and my interactions with her were mostly on social occasions with Hamburger. Like Hamburger, I had never had much interest in studies at the molecular level; among other reasons, my brief but dismal experience with neuropharmacology research as a med student left a lingering bad taste, and, with occasional striking exceptions (such as the discovery of endorphins in the 1970s), I felt that many molecular studies were revealing more and more about less and less. Nerve growth factor was another exception. Not only did this agent promote the survival of the very neurons we were studying, but it also influenced the growth of the axonal and dendritic processes of the classes of neurons that were sensitive to it and, by implication, the synaptic contacts they made (see Figure 5.3). It was not much of a stretch to imagine that competition for and acquisition of such factors was the basis of the maintenance of synaptic connections we had been providing evidence for, and that this "trophic theory" of how synapses were regulated in the nervous system was a general rule. The idea was that each class of cells in a neural pathway was supporting and regulating the connections it received by trophic interactions with the cells it innervated down the line, resulting in a coordinated chain of connectivity that extended from the periphery centrally to the spinal cord and, ultimately, on through the controlling centers in the brain ( Figure 5.5).

Figure 5.5 Scheme of synaptic maintenance that could enable the coordination of synaptic connectivity throughout an entire neural pathway (NGF stands for nerve growth factor). (Reprinted by permission of the publisher from Body and Brain: A Trophic Theory of Neural Connections by Dale Purves, p. 135, Cambridge, Mass: Harvard University Press, Copyright © 1988 by the President and Fellows of Harvard College. After Purves, 1986.)

Brain Pathways Jeffrey Fortuna

Peripheral sympathetic tar^ei

The goal of this work on synaptic connectivity in mammals was not to sort out what molecules might be involved (the paradigm provided by nerve growth factor was sufficient evidence, and many labs were studying this agent by the mid-1970s), but instead to determine the governing principles. Jeff Lichtman was the prime mover in pursuing this aim. My initial doubts about taking him on as an M.D./Ph.D. student had been quickly dispelled. Within a few weeks, it was obvious that Lichtman was extremely bright, and even though he was a student 13 years my junior, we were soon discussing things as colleagues. Persistence is another good quality for a scientist to have, and Lichtman was as tenacious and determined as he was smart. Shortly after he started working in the lab, I found him underneath one of the cabinets removing the drain trap with a wrench. He had stained some neurons in a ganglion with a new dye technique, which had accidentally gone down the sink during the procedure. Although the missing ganglion was only as big the head of a pin, Jeff eventually found it in the muck that he pulled out of the trap.

The main problems that concerned us for the next several years were the nature of competition among the axons that innervate target nerve cells, and how the signaling activity of competing nerve cells affects the balance of synaptic connectivity (a problem related to the effects of activity that Bert Sakmann and I had wrestled with in London). We were convinced that nerve cells and their targets must interact in sorting out the connectivity of functioning circuits in much the same way that elements in an ecosystem eventually establish equilibrium as they compete for limited resources. This idea about neural connectivity was not new—the Spanish neuroanatomist Ramon y Cajal had written in flowery prose about this ecological concept of neural development in the late nineteenth century—but no one had determined the neurobiology of how this competition might actually work.

The closest anyone had come to directly exploring the issue of synaptic competition by the mid-1970s was Michael Brown, David Van Essen, and Jan Jansen studying the developing innervation of skeletal muscle fibers. I didn't know Brown, but Van Essen had been a graduate student at Harvard when I was there (he joined John Nicholls's lab for his doctoral work about the time I left to work with Jack McMahan), and Jansen had worked in Nicholls's lab when on sabbatical from his position in Oslo that same year. A few years later in Oslo, they had shown that during the first few weeks of postnatal life, each fiber in a rat muscle is contacted by more nerve terminals from different axons than persist in maturity (Figure 5.6A), providing another clue about the nature of synaptic competition and maintenance. A natural question was whether the innervation of neurons followed the same rules as muscle fibers, and Lichtman's thesis work showed that it did ( Figure 5.6B).

Figure 5.6 The competitive interaction between axon terminals for the innervation of target cells during synaptic development. A) The elimination of all synapses except those made by a single axon on each developing muscle fiber during the course of early postnatal life. B) The analogous phenomenon on maturing neurons (in this case, on a class of autonomic ganglion cells that lack dendrites, making the analysis much simpler). (Adapted from Purves, Augustine, et al., 2008)

A Muscle cells B GiLiiglioti cells

Figure 5.6 The competitive interaction between axon terminals for the innervation of target cells during synaptic development. A) The elimination of all synapses except those made by a single axon on each developing muscle fiber during the course of early postnatal life. B) The analogous phenomenon on maturing neurons (in this case, on a class of autonomic ganglion cells that lack dendrites, making the analysis much simpler). (Adapted from Purves, Augustine, et al., 2008)

A Muscle cells B GiLiiglioti cells

Understanding the interactions among axon terminals and the synapses they make on target cells remains woefully incomplete today. However, some important principles emerged from work that Lichtman and several other fellows carried out in the lab over the next few years. One principle is that the spatial configuration of a neuron is a critical determinant of the innervation it receives. For nerve cells without dendritic processes, such as those in Figure

5.6B, the end result of the initial competition is innervation by many synaptic endings that all arise from the same nerve cell axon. If target nerve cells have dendrites, however, the number of innervating axons increases in proportion to the number and complexity of these branches ( Figure 5.7A). Moreover, after a given axon makes some synapses on a target neuron, the axon is somehow informed by the conjoint activity of the pre- and postsynaptic neurons that the target cell is a favored site for the elaboration of additional synaptic endings ( Figure 5.7B). This focusing of synapses occurs despite the presence of numerous other valid target neurons in the immediate vicinity. Therefore, the synaptic terminals made on a target neuron act as sets instead of individual entities during the establishment of neural circuits. This and much other evidence implies that neural activity—action potentials that lead to transmitter release at the synapses in question—is somehow involved in circuit formation. Our initial ideas about competition for limiting amounts of target-derived signaling agents is, in retrospect, only part of a more complex story, as shown by Lichtman's remarkable work on this issue during the last 35 years and counting.

Although these observations were intriguing, it was increasingly clear that understanding what was going on during the formation and maintenance of synapses required a way to directly follow the progress of synaptic contacts on the same nerve or muscle target cell over periods of days, weeks, months, or longer. This goal seemed technically feasible in the peripheral nervous system, and would allow us to watch how competition operated during development, and how synaptic connections continued to be modified in maturity. Because encoding experience during life depends on functional and anatomical changes in neural connectivity, the expectation was that synaptic connections would gradually change over time and that we would be able to witness the process in action. The next step was thus to figure out how to monitor the same synapses chronically.

Our first stab at this was indirect, based on the ability to identify the same neuron in the autonomic ganglia of a living animal on different occasions. Given that each neuronal cell body has a somewhat different appearance in the cobblestone-like pattern of cells visible on the surface of a ganglion (Figure 5.8A), it is not hard to find the same neuron during an initial surgical exposure and at a second such operation after an arbitrarily long interval. We could inject an identified neuron with a nontoxic dye and photograph the configuration of its dendrites. By carrying out the same procedure weeks or months later, we could determine how much, if at all, the dendritic branches changed during the interval. Because the dendrites of ganglion neurons are studded with synapses, any change in the architecture of the dendritic branches would imply ongoing changes in synaptic connectivity. These studies showed that dendrites are slowly being remodeled, and therefore that the synaptic connectivity of the neurons must be slowly changing as well ( Figure 5.8B).

Figure 5.7 The dependence of neuronal innervation on the geometry of the target cells. A) The proportionality between dendritic complexity and convergence: The more dendrites a neuron has, the greater the number of different nerve cell axons that innervate it, thus affecting the integration of information. B) The synapses arising from a single axon act as a set during innervation. In this example, a single axon leading to the ganglion has been injected with a dye; each cluster represents a group of synapses made on a particular nerve cell in the ganglion (the outline of the ganglion is shown). Despite hundreds of available target neurons, the labeled axon makes many synapses on just a few target cells. (A is after Hume and Purves, 1981; B is from Hume and Purves, 1983)

Figure 5.8 Ongoing changes in the synaptic connectivity of nerve cells. A) The appearance of the surface of an autonomic ganglion in the same mouse at an interval of several months; the pattern of cells enables the identification of individual neurons (for example, the one marked with an asterisk) after an arbitrary interval. B) Examples of differences in the configuration of selected portions of the dendrites arising from the same neuron during the interval indicated. Open arrowheads indicate the loss of a dendritic process, and filled ones indicate the addition. (After Purves et al., 1986)

Figure 5.8 Ongoing changes in the synaptic connectivity of nerve cells. A) The appearance of the surface of an autonomic ganglion in the same mouse at an interval of several months; the pattern of cells enables the identification of individual neurons (for example, the one marked with an asterisk) after an arbitrary interval. B) Examples of differences in the configuration of selected portions of the dendrites arising from the same neuron during the interval indicated. Open arrowheads indicate the loss of a dendritic process, and filled ones indicate the addition. (After Purves et al., 1986)

Monitoring the synaptic endings themselves over time would be more revealing, and this is what we set out to do next. The problem in this project was that, unlike the cell bodies in Figure 5.8, synapses are far too small to be directly and routinely injected with a dye. To visualize synapses, we needed a dye that the terminals would quickly absorb when they were bathed in it that would then diffuse away and not damage the endings. Finding such a reagent was a matter of trial and error, and the person who undertook this thankless task was Lorenzo Magrassi, a smart and hard-working medical student from

Italy who had come to spend a year in the lab in 1985. Magrassi, who knew a lot about chemistry, applied one plausible reagent after another to synaptic endings on mouse muscles in a dish while he observed the results under a microscope. After many weeks, he finally succeeded in finding a dye that met the criteria. Lichtman (who was by then a faculty member), Magrassi, and I used this approach to watch the same synapses on muscle fibers over months by finding and restaining the same synaptic endings ( Figure 5.9). The method also worked for the synaptic endings on identified ganglion cells and synapses on neurons that could be similarly followed over time. In both cases, synaptic terminals gradually changed, slowly on mature muscle fibers and faster on neurons.

Figure 5.9 Observing synapses directly over time. A) Exposure of an accessible muscle in the neck of an anesthetized mouse. All the synaptic endings on the muscle fibers were then stained by the application of a nontoxic fluorescent dye. B) The same synaptic endings on a single identified muscle fiber (see Figure 5.6A) after an interval of several months, showing small but definite changes. (Reprinted by permission of the publisher from Body and Brain: A Trophic Theory of Neural Connections by Dale Purves, p. 111, Cambridge, Mass: Harvard University Press, Copyright © 1988 by the President and Fellows of Harvard College. After Purves, 1986.)

From most perspectives, this effort to understand the formation and maintenance of synapses had been quite successful. But by the mid-1980s, after I had been laboring away on these issues for more than ten years, I didn't think the research was going so well. The reasons were several and were only partly caused by science. Perhaps it is more accurate to say that science proceeds like any other human enterprise, with real and imagined factors determining the mindset of any individual practitioner. With respect to the science, it was not clear at that point what to do next. Directly monitoring synaptic change in muscle fibers and ganglia had been a fine way to start, but no one was going to get very excited about this work if similar studies of synaptic stability in the brain could not extend it. After all, the brain determined behavior and the cognitive processes that I and everyone else ultimately wanted to understand. I had viewed these studies of synapses in ganglia and muscle as simple model systems for understanding what was likely happening in more interesting parts of the nervous system. But the techniques we had been using were difficult enough to apply in the peripheral nervous system, and, for various reasons, they were impossible to apply to synapses in the brain. Within a decade, further advances in molecular biological methods resolved this impasse by providing labels that could be introduced into neurons by gene transfection. This methodology eventually enabled Lichtman and his collaborators and many others to begin tackling these types of problems in the brain, but that possibility was not on the horizon in the mid-1980s.

Other factors were at work as well. Hunt, the head of the department in which I had worked for most of this time, had retired and moved to France. The department's focus had changed to cell biology and, as a result, I (along with Lichtman and Sanes) had moved to the Department of Anatomy and Neurobiology in 1986. Gerry Fischbach was by then running the department because Cowan had also left to take a position as the chief scientific administrator at the Howard Hughes Medical Institute. Fishbach is a lovely person and was a fine chairman. But he was a peer, creating a situation quite different from the paternal figure Hunt had been. In the new department, I was among colleagues who were explicitly interested in the brain and had a different outlook on neuroscience than the one I had grown up with at Harvard and University College, and that I had continued to experience in Hunt's department. Finally, my relationship with Lichtman became increasingly awkward. He was a faculty member with his own very successful lab, and although we had continued to collaborate, the relationship was no longer the one that I had enjoyed for many years. We were now working on the same issues and, to some degree, had become competitors; the situation was delicate for both of us.

In a couple years, I would be 50, and the undeniable fact of middle age combined with these several circumstances combined to trigger another bout of depression, this one more serious than those I had experienced as a teenager, in college, or in med school. Although I kept coming to work every day, my enthusiasm for what I was doing dwindled. I saw a psychiatrist, who started me on an antidepressant, and when the drug he prescribed didn't work (ironically, it was the one I had worked on in my summer of neuropharmacology research as a med student), the depression deepened. Mainly as a result of my wife's support, the counsel of another psychiatrist, a different medication, and perhaps just the passage of time, I gradually began to see a plausible future again. After all, I still would presumably have more years left in neuroscience at age 50 than the number I had already completed.

Even so, I realized that plugging away on the same issues in the peripheral nervous system would not suffice. Having gradually returned to a better frame of mind, I began to feel that I had achieved enough success to take some bigger scientific chances. I had started out with broad philosophical interests in the brain, but by virtue of my training, the people and the science that I admired, and the overall direction of neuroscience, I was de facto a reductionist. I had worked on some important issues in model systems, but these would never be more than indirectly related to the things that had first interested me about the brain. With perhaps another 20 years or so to go, I felt that I owed it to myself to at least think about doing something that might go beyond the conventional framework that I had assiduously learned, worked within, and taught for the previous two decades. As it had been ever since those inconclusive evening meetings at Harvard where we postdocs tried to come up with a list of challenges for the future, it was difficult to identify a significant problem. And although I was now a senior figure in the field, when I poked my head up and tried to look beyond the boundaries of the mainstream neuroscience I had been practicing, I had no idea what sort of issue to take on next.

Was this article helpful?

0 0
Peripheral Neuropathy Natural Treatment Options

Peripheral Neuropathy Natural Treatment Options

This guide will help millions of people understand this condition so that they can take control of their lives and make informed decisions. The ebook covers information on a vast number of different types of neuropathy. In addition, it will be a useful resource for their families, caregivers, and health care providers.

Get My Free Ebook


Post a comment