Good luck, second chances and being a second choice
As told by Oswald Steward

In dendrites, lie the sites where memories spawn
Near the structures towards which one’s eyes are drawn
Small spots that line the message plain to see
Beneath the synapse, proteins soon to be
Quatrain by Oswald Steward

Looking back at my life, I’m astounded at the many instances where good luck was the only reason my life did not go in a very different way. I lived in a time and place where second chances allowed mis-steps to be overcome.

I was born in Bermuda. My parents moved to Bermuda after WWII where my father was a firefighter at the Bermuda U.S. Naval base. My family moved to Cocoa Florida when I was 2 years old, where my father continued as a firefighter at Patrick Air Force Base near Cape Canaveral.

Living in Cocoa in this incredible era forged my attitude that science equals adventure.

My early childhood in Cocoa was during the pre-Sputnik years. There was great excitement about the USA being the first into space. We watched early rocket launches from our back yard--exciting because many malfunctioned and were “terminated” in spectacular explosions. I remained in Cocoa through John Glenn’s orbital mission in 1962. Living in Cocoa in this incredible era forged my attitude that science equals adventure.

Early interest in Biology
My childhood home was a post-war tract house built on previously swampy ground, which was drained by digging drainage ditches. The ditches attracted a variety of Florida water fauna—minnows, turtles, snakes (some poisonous) and juvenile alligators that were fortunately too small to do any real harm to a curious boy. I spent my time trying to capture turtles and snakes to bring home to aquaria. I studied books to learn how to identify different types of snakes—important knowledge if you were going to bring some home.

My mother emphasized my academic development. She was my champion when I made bad decisions. My father taught me to build things and to have less fear than I should.

My mother attended college for 2 years. She didn’t pursue career aspirations when I was young but at age 50, went back to get a BS in Speech Therapy to start a new career. She emphasized my academic development, tolerated the various creatures I brought home, and was my champion when I made bad decisions. My father might have attended college but contracted tuberculosis, spent 8 months in a TB sanitarium and then had to join the workforce. He fostered my learning by bringing interesting things home from the air force base—things that burned intensely or blew up, asbestos gloves—"look son, you can reach into the fire”, a bottle of mercury to investigate properties of liquid metal. The latter may explain some of my quirks. My father taught me to build things and have less fear than I should about trying to fix things, from electrical to replacing faulty vacuum tubes in TVs. This early experience helps me maintain scientific equipment far beyond its useful life.

If my parents were alive today to hear about The Kavli Prize, my father would be proud; my mother would believe it

Chemistry sets for birthdays
Birthday presents included chemistry sets to make a mess, and a kid’s microscope through which I saw live protozoa from the ditch. Growing up, conversations were that I should go to college, but an advanced degree was not imagined. If my parents were alive today to hear about the Kavli Prize, my father would be proud; my mother would believe it.

Public schools in Florida were segregated in the 1950’s, but schools in Cocoa were better than the Florida average (at least for white kids). My school offered math and science courses in early grades; I took the courses, but did not apply myself, and my grades were average.

I were the first to be suspended from Boulder High School for long hair

Our family moved to Boulder Colorado in 1962, home of the University of Colorado. This was a huge culture shift, but my lack of interest in academics continued. As I started high school, I found more interesting things to do than go to class regularly. The predictable result was D’s and Fs. My only distinction in my first year was that a friend and I were the first to be suspended from Boulder High School for long hair.

Course correction
Starting my second year in high school, I met my future wife Kathy in the Biology class I had to re-take. I helped her dissect her frog; then we had our first date. Suddenly, I had a reason to attend class! One evening, she told her parents that I was picking her up to go to the library to study. I picked her up, and asked “what do you want to do?” She said, “go to the library of course.” I wasn’t prepared but found something to read while she actually studied. Next time, I did some homework and discovered that class was more enjoyable.

Conversations with Kathy’s smart friends who were children of CU professors sparked my intellectual interests. I enjoyed classes in Political History, Sociology, Anthropology, and especially two elective courses (Anatomy and Psychology) both taught by Mr. Robert Hamilton, who’s dry wit I enjoyed. My grades improved greatly and I graduated from high school one year late, albeit in the lower 10% of my graduating class because of the mis-spent first year.

Second chance
As my academic performance improved, attending college became a goal. Kathy had a scholarship for music school at the University of Colorado, but CU seemed out of reach for me. A second chance came because my high school counselor looked past my early misadventures and recommended me for a trial program at the University of Colorado that allowed 20 students who seemed promising but had issues to take Freshman English and 2 other courses during the summer session. If the student earned grades of “B” or above in all courses, they were admitted to CU. I received A’s, and Kathy and I were on our way to bachelor’s degrees at CU. I am forever indebted to my advisor, Mr. Peterson, for this second chance!

As a freshman, I had part-time jobs as a night janitor at a bank and cashier in a “head shop” that sold marijuana paraphernalia and psychedelic posters

We received some financial help from our family during our undergraduate years, but we still had to work, which was fortunate. As a freshman, I had part-time jobs as a night janitor at a bank and cashier in a “head shop” that sold marijuana paraphernalia and psychedelic posters. Then, in the first semester of my sophomore year, I took a class called “Biological Psychology” from Professor Kurt Schlessinger. I was completely enthralled and applied to work in his lab cleaning cages in the mouse colony. Kurt had been at the University of North Carolina and was involved in studies in the late 1960’s on RNA changes during learning. The underlying idea was that memory in the brain might use the same molecules as genetic memory. Kurt continued studies in this vein. His senior graduate, Linda Uphouse, was studying changes in DNA complexity with learning in mice (a measure of the opening up of chromatin for transcription). Linda helped me advance from cleaning cages to helping carry out experiments. I loved planning and executing experiments and then going to the next and decided that a career as an academic scientist was my goal. I enthusiastically took extra classes to complete my BA in 3 years.

Looking toward graduate school, I was fascinated by studies reporting that disruption of protein synthesis after a learning experience impaired memory. These approaches blended concepts and techniques of Molecular Biology and Physiological Psychology and I decided that exploring the role of protein synthesis in memory was the question I wanted to pursue. I applied to several graduate programs, but my dream was to be admitted to the new Department of Psychobiology at the University of California Irvine (UCI).

I felt relief but also guilt because I was able to continue my education whereas others went to Vietnam; some didn’t return

But there was one issue - it was 1969 and I was subject to the military draft. The order of callup was determined by a lottery. We watched the televised lottery with hope and apprehension as birthdays were drawn out of a tumbling bin. My birthday came up #243. I felt relief but also guilt because by the luck of the draw, I was able to continue my education whereas others I knew went to Vietnam; some didn’t return.

Rejected at first
I was disappointed to receive a rejection letter from UCI in March 1970. I began to make alternate plans, but in April, I received a call from Carl Cotman, a new assistant professor at UCI, telling me that some of their first choices had declined, and he offered admission into his lab. Being a second choice was fine with me, and I immediately accepted.

Carl was a protein biochemist. His first grant was to characterize proteins in the postsynaptic junction of CNS synapses. When I joined the lab, Carl was extending his studies to the biochemistry of learning and memory. My first project was to assess whether synaptic activity enhanced postsynaptic protein synthesis. For this, we collected Aplysia from nearby tide pools, dissected their abdominal ganglia with attached nerves, placed the ganglia in media with radioactive amino acids and stimulated the nerves. I dissected the large R60 neuron that receives input from the stimulated nerve and assessed incorporation into protein by scintillation counting. Results were inconclusive, but going to the tide pools was definitely fun.

Refuted Cajal'a dogma
By the end of my first year, something new began to explode. Geoff Raisman had published a paper reporting synapse growth after lesions in the mature rat brain (sprouting). This report shook the foundation of Cajal’s dogma that “..nerve paths in the brain are fixed and immutable: everything may die, nothing may be regenerated“.

Inspired by the potential implications of synapse growth in the mature brain, Carl teamed up with Gary Lynch, another new faculty member at UCI, to test whether sprouting occurred in the hippocampus, a structure with highly laminated inputs. The approach was to lesion the main input to the hippocampus from the entorhinal cortex and use histochemistry for acetylcholinesterase (AChE) to detect potential sprouting of cholinergic septo-hippocampal axons. The results were striking—dramatic increases in staining for AChE in the denervated lamina indicating sprouting.

Eureka for dissertation project
My second-year project was to use a physiology setup in Gary’s lab to test whether sprouted septal cholinergic axons were physiologically functional. I learned the basics of in vivo neurophysiology, but results were confusing because the cholinergic system operated through metabotropic receptors and did not generate fast EPSPs. One evening, while reading a reprint after dinner, the idea came to me that with a unilateral lesion, the most appropriate source of replacement connections would be from the contralateral entorhinal cortex. Having rats with entorhinal cortex lesions, I drove back to lab and did the first physiology study to test this. By 1:00AM, I had clear evidence of a functional crossed pathway. I excitedly drove home, knowing I had a potential dissertation project. It was a huge gift that I had the freedom to carry out this project independently and to take the project with me to my first faculty position.

At that time it was sometimes possible to get a faculty position directly after a PhD. The Department of Neurosurgery at the University of Virginia (UVA) was recruiting a basic scientist, and I was offered the position.

Familiy life as a young scientist
We moved to Charlottesville in July 1974, I wrote my first NIH grant and our first child Jessie was born in December. Over the next 2 years, Kathy completed her doctorate in special education at UVA, our second child Oswald (Ossie) was born in 1978, and our family was complete. It was a whirlwind, but it was a great time to be a young scientist!

The first years were spent on anatomical studies characterizing the process of reinnervation and behavior studies showing that sprouting contributed to recovery of function. Then, I began studies to try to define molecular mechanisms. I decided that a first step would be to test whether synapse growth required increases in protein synthesis by pre- or postsynaptic neurons.

Images triggers new insight
To assess this, Barry Fass, a postdoc who had just joined the lab, injected radioactive amino acid at different times after lesions in rats and prepared brain sections for autoradiography. Based on the prevailing dogma at the time, we expected to see increases in protein synthesis in neuronal cell bodies. Instead, we saw increases in protein synthesis in dendritic laminae of the neurons being reinnervated. My first thought was that this was due to increased protein synthesis by glial cells. We had been carrying out electron microscopic studies of synapse replacement, so I asked my technician to collect images through the dendritic laminate, expecting to see ribosomes in glia. When I first looked at the micrographs, what stood out were striking images of polyribosomes in dendritic spines. The images triggered an “aha”moment--these polyribosomes could be making proteins for the synapse!

A-hah-moments don’t occur in a vacuum—they certainly depend on background knowledge into which a new observation fits. Colleagues and intellectual environment are important for testing and honing the ideas

The first use of "Hebb-like" synapses
At the time of my discovery of synaptic polyribosomes, William B. (Chip) Levy and I were also carrying out studies on associative interactions between synapses during induction of long-term potentiation (LTP). Chip gave up a faculty position to come to UVA to collaborate on studies using the anatomical system I had characterized—the sparse crossed projection from the entorhinal cortex to the dentate gyrus. The sparse crossed and main ipsilateral pathway converge on dentate granule cells, and Chip had the inspiration that this would be a great model to test whether LTP could be induced in the weak crossed pathway by pairing its activation with the strong ipsilateral pathway. This is a property predicted for synapses that operate on the basis of the Hebb principle. The answer was yes. To my knowledge, our first paper on this was the first to use the term “Hebb Synapse” (actually, we said “Hebb-like synapse”). Thus, I was already thinking about mechanisms of synaptic plasticity when I saw the polyribosomes at synapses. Chip loved to talk science, which we did on daily walks from our lab to the swimming pool for noon work-outs.

The "good enemy" challenged the thinking
The other close friend and colleague was Ed Rubel who was studying the development of auditory pathways in post-hatch chickens, especially how synaptic activity regulated dendritic growth and cell survival. He discovered that removal of the cochlea led to cessation of protein synthesis in neurons in the cochlear nucleus leading to their death—another link between synaptic activity and protein synthesis. Ed was a “good enemy”, who challenged my thinking. Our families vacationed together at beach and mountains for skiing and hosted backyard barbecues for our labs (dig a pit, build a low fire, add pork roast or sometimes a whole pig, tend the fire overnight while we played poker).

The rich discussions with these two amazing colleagues helped hone my ideas regarding protein synthesis at synapses and it’s potential role in synapse growth and plasticity.

I continue to update the diary today

I have a record of the evolution of my thinking because I started a science diary, which starts with: “I’m starting this diary because I believe that the polyribosome story is going to evolve into a very important story. I have the feeling that it may be the most important work I’ve done to date”. I continue to update the diary today.

I had some misgivings and so reached out for advice. First, I went to the American Association of Anatomists meeting in New Orleans to find Alan Peters, first author of the book “Fine Structure of the Nervous System”; we sat together looking at my micrographs. He confirmed my interpretations-- these were polyribosomes and the clusters suggested that the ribosomes were on an mRNA molecule actively engaged in protein synthesis.

Exchanging letters with Eccles
Second, I wrote Nobel Laureate John Eccles, who I had met when I hosted his visit for the neuroscience seminar series at the University of Virginia. I sent photomicrographs and explained my ideas. He wrote back a hand-written letter dated 30 Nov 1981: I am very ready to accept your idea that the polyribosomes that you find in the dendrite concentrated at the origin of spines are related to protein synthesis in the spines. I congratulate you on this fine work and hope to hear from you about the effect of synaptic activity on polyribosome concentration.

There followed an exchange of letters, which I kept, that boosted my confidence that I was onto an important story

Testing
Buoyed by these confirmations and encouragement, I launched studies to: 1) test hypothesis about the role of synaptic protein synthesis in adult synaptogenesis after injury and in development; 2) identify proteins synthesized at synaptic polyribosomes.

Over the next 5 years, our electron microscopic studies established that polyribosomes were more abundant during both developmental synaptogenesis and during reinnervation. Graduate student Anuradha (Anu) Rao obtained the first evidence that one of the proteins synthesized at synapses was the alpha-subunit of calcium calmodulin dependent kinase II (alpha CAMKII). For this, she isolated “synapto-dendrosomes” (pinched off nerve endings with attached dendritic fragments) incubated these with radioactive amino acid protein precursors, then used western blot autoradiography to determine the molecular weights of the newly-synthesized proteins. A protein at the molecular weight of CAMKII was the most heavily labeled. Anu went on to become Senior Editor at the journal Neuron from 2001-2004. Sadly, Anu suffered a fatal medical emergency while flying home from the Society for Neuroscience meeting in San Diego in 2004.

Visualizing the dendritic tree
Our progress over the next decade was slow, but was greatly accelerated when I recruited Gary Banker to our new Department of Neuroscience at UVA. Gary had developed the dissociated hippocampal neuron culture system in which neurons are grown on glass coverslips rather than on a bed of glial cells. With these, the dendritic tree and axon arbor of individual neurons could be visualized.

These beautiful cultures made it possible to define characteristics of RNA transport into dendrites. Graduate student Lauren Davis defined the time course of transport of newly-synthesized RNA into dendrites by pulse-labeling cultures with radioactive uridine and preparing neurons for autoradiography at different times after labeling. Newly-synthesized RNA moved into the dendrites at a relatively slow rate, reaching the ends of dendrites after about 24 hours.

We later realized that this technique was mainly measuring newly synthesized ribosomal RNA, and likely the transport of newly synthesized ribosomes. Graduate student Robin Kleiman deployed the newly-developed technique of in situ hybridization to demonstrate that the mRNAs for alpha-CAMKII and the microtubule-associated protein MAP2 were present throughout dendrites in hippocampal neurons whereas mRNAs for cytoskeletal proteins including tubulin and neurofilament were restricted to cell bodies. Enrique Torre, a postdoctoral fellow from Cordoba, Argentina used antibodies against protein constituents of the endoplasmic reticulum (ER) and Golgi apparatus (glycosyl transferases) to demonstrate ER and Golgi-like outposts in dendrites. Pulse-labeling with different sugars demonstrated active protein glycosylation in dendrites. These studies indicated Golgi-like capability for local glycosylation in dendrites. Taken together, these studies revealed that all the machinery necessary for protein synthesis and processing was present in dendrites.

The size of the audience didn’t bother me – but then I saw Eric Kandel (Nobel Prize Laureate 2000), who always sits in the front row

Not much "push-back", but also not much "hold"
I’m often asked whether our story on a distributed network of protein synthetic machinery was controversial given the dogma at the time that all neuronal protein synthesis occurred in the cell body. There wasn’t much active pushback, but the idea didn’t really take hold for about a decade. I didn’t realize the extent of interest until 1993, when I gave a Special Lecture at the Society for Neuroscience meeting. There were over 2000 people in the audience. The size of the audience didn’t bother me – after the lights went down, I couldn’t see beyond the first 2 rows – but then I saw Eric Kandel (Nobel Prize Laureate 2000), who always sits in the front row.

By the early 1990’s, it was increasingly apparent that RNA localization and local protein synthesis operated in other complex cell types and in embryogenesis. Cell biologist Rob Singer had established this in migrating fibroblasts. I met Rob at a FASEB conference on the neuronal cytoskeleton in 1992 and we hatched a plan to organize a FASEB conference on RNA localization and local protein synthesis in different cell types and developing organisms. The first meeting was in Snowmass, Colorado in 1994 and drew about 40 participants. Interactions were excellent, and the meeting expanded, continuing today as Gordon and EMBO Conferences on alternate years.

Wanted back to the UCI
In the early 1990s, I felt an increasing responsibility to devote some on my research to un-met medical needs, and decided to explicitly extend my research to the problem of spinal cord injury. This research program progressed in parallel with our research on RNA localization, and led to my being recruited back to the University of California Irvine in 1999 as founding Director of the Reeve-Irvine Research Center.

In Paul Worley’s words, Arc became a Rosetta Stone for deciphering principles of RNA transport and localization at synapses during synaptic plasticity

A paradigm shift in 1995
A major conceptual shift in our understanding of dendritic mRNAs was launched by a phone call from Paul Worley at Johns Hopkins University in 1995. Paul had completed a study to identify new immediate early genes (IEGs) induced by synaptic activity and discovered one whose mRNA moved into dendrites. He called it activity regulated, cytoskeleton-associated protein (Arc). He asked if I would be interested in studying this novel gene, and of course, I said yes immediately.

In Paul Worley’s words, Arc became a Rosetta Stone for deciphering principles of RNA transport and localization at synapses during synaptic plasticity. Postdoctoral fellow Chris Wallace demonstrated that after a seizure, newly synthesized Arc mRNA was transported from the nucleus throughout dendrites within about 30 minutes whereas mRNAs for other IEGs remained in the cell body. Differential sorting was maintained when protein synthesis was blocked, indicating that selective transport into dendrites was based on a signal in Arc mRNA itself (zipcode).

A remarkable finding came when we assessed Arc localization with stimulation of input synapses to induce LTP. As Arc mRNA moved into dendrites, it localized selectively in dendritic domains contacted by active synapses. Using this model, we then used pharmacological interventions to determine that the signal for Arc localization at active synapses was created by NMDA receptor activation—the same mechanism that underlies induction of LTP.

The importance of graduate students
The story was further advanced by the research of 3 fabulous graduate students.

Fen Huang characterized features of the mechanism for docking Arc mRNA at active synapses, showing that disruption of Actin polymerization disrupted Arc mRNA localization.

Patricia Salgado Pirbhoy demonstrated that synaptic activity triggered phosphorylation of ribosomal protein S6 in exactly the portion of the dendrite in which Arc mRNA localizes suggesting a mechanism for translational regulation.

Shannon Farris established that Arc mRNA at synapses is degraded in an activity and protein synthesis dependent fashion, revealing a mechanism for down-regulation of Arc protein expression at synapses. Shannon’s research during postdoctoral studies with Serena Dudek and in her own lab at Virginia Tech University established that different populations of neurons in the hippocampus have a different compliment of dendritic mRNAs.

Patricia Pirbhoy has returned to our group and is currently using spatial transcriptomics to identify new members of the family of mRNAs that increase in abundance in dendrites in response to activity.

My lab’s research now represents a small fraction of the amazing work being done by laboratories throughout the world on RNA localization and local translation. I’m thrilled to continue to be a part of the expanding story.

Retrospective
I feel okay about my work-life balance. Kathy and I coordinated schedules as she pursued her career in special education in the public school system. We lived close to the university, so I came home for dinner and worked together to put the kids to bed, then I often went back to the lab. We took family vacations in Colorado mountains for camping and backpacking and took our kids out of school for skiing/snowboarding at the Winter Conference on Learning and Memory and Winter Conference on Brain Research. We took time off to attend my daughter’s horse shows and my son’s swim meets; many entries in my science diary were written at these events.

I cannot thank my wife Kathy enough for over 60 years of love and support—and tolerance of my life of a scientist. Our kids matured into amazing, responsible people despite missing a few days of school for Steward family trips; grandchildren are prospering and are taking time off from work and school to attend the Kavli award ceremony. I can’t imagine a life more fulfilling than life as a scientist.