This is an obituary for Eugene Roberts (1920-2016), an outstanding neurobiochemist who was the first to report on the discovery of gamma-aminobutyric acid (GABA) in the brain, and whose research focused on the important role of GABA in human health and disease. Dr. Eugene Roberts This is an obituary for Eugene Roberts (1920-2016), an outstanding neurobiochemist who was the first to report on the discovery of gamma-aminobutyric acid (GABA) in the brain, and whose research focused on the important role of GABA in human health and disease. Dr. Eugene Roberts, a true visionary and pioneer of neurobiochemistry and neuroscience and distinguished contributor to the Journal of Neurochemistry (for instance, his well recognized paper on L-glutamate decarboxylase, Wu and Roberts, 1974), passed away on November 8, 2016 at the age of 96. Eugene discovered the presence of unusually large amounts of γ-aminobutyric acid (GABA) in the brain in 1949. This historic observation led to his major career focus on the important roles of GABA in human health and disease, such as in epilepsy, schizophrenia, Huntington's disease, Alzheimer's disease, autism, drug addiction, and mental retardation. Eugene's vision led to a better understanding and treatments for these important clinical problems. He recognized that understanding GABA's importance would require a visualization at the light and electron-microscopic levels of the neurons that synthesize and liberate the substance. This had never been achieved before for any neurotransmitter and would require collaboration. He established a Division of Neurosciences at the City of Hope National Medical Center in Duarte, California, in 1968, one of the first such organizations in the world. Some of us were fortunate enough to join Eugene in the early phase of his pursuit of GABA's functional importance. We vividly remember the excitement when purifying the GABA synthesizing enzyme, L-glutamate decarboxylase (GAD), the GABA degradation enzyme GABA transaminase (GABA-T) and the development of specific antibodies to GAD and GABA-T allowing visualization of GABAergic neurons by newly developed immunohistochemical techniques. This was a truly interdisciplinary effort driven by Eugene's dream to ‘see’ GABA neurons. The initial group included J-Y. Wu, A. Schousboe, T. Matsuda, K. Saito, R.P. Barber, J.E. Vaughn, J.G. Wood, and B.J. McLaughlin. A highlight of that time was a daily brown bag lunch around the picnic table outside the lab building where we listened to Eugene's personal views about what was important in science. His views were always provocative and challenging and he taught us all how to think about the ‘big picture’. For example, one of Eugene's favorite ideas at the time was that the nervous system is tonically inhibited by the wide distribution of inhibitory GABA neurons and synapses and only released from this state when circuits are appropriately stimulated. This then revolutionary concept of ‘disinhibition’ is now widely accepted in neuroscience. Important milestones in the early history of GABA were originally presented at the first conference devoted entirely to this molecule organized by Eugene in 1959, at City of Hope and documented in the proceedings published in 1960 (Roberts et al. 1960). Subsequent work including identification of GABAergic neurons and circuits, GABA synthesis and removal, characterization of GABA receptors and transporters were presented at a workshop, again organized by Eugene, in 1975 held at the J & R Double Arch Ranch in California's Santa Ynez Valley, a venue owned by Ray A. Kroc, founder of McDonalds as well as The Kroc Foundation. The proceedings were published in 1976 (Roberts et al. 1976). Born Evgeny Rabinowitch in Krasnodar, Russia, Eugene was brought by his parents to Detroit Michigan, in 1922. In 1936 he received a B.S. degree in chemistry, magna cum laude from Wayne State University. A scholarship from the McGregor Foundation and a University Fellowship enabled him to earn M.S. and Ph.D. degrees in 1941 and 1943, respectively, at the University of Michigan. He spent his early career working on the Manhattan Project's inhalation section at the University of Rochester investigating the toxicology of uranium dusts. In 1946, he joined the Division of Cancer Research at Washington University in St. Louis where he studied nitrogen metabolism in normal and neoplastic tissues and was one of the first to receive a grant from the newly organized National Cancer Institute of the National Institutes of Health. In 1954, he joined the staff of the City of Hope Medical Center in Duarte, California as Chairman of Biochemistry and Associate Director of Research at a time of its transition from a tuberculosis sanatorium to a modern biomedical and cancer research institute. He convinced the institutional leaders that the best way to organize a successful research effort would be to hire talented and committed young scientists and allow them the freedom to seek their own unique destinies while furnishing moral and material support. This approach produced six members of the National Academy of Science, including Eugene himself, a remarkable record for such a small research institute. Eugene played an important role in the organization of the American Society for Neurochemistry serving as a counselor (1970–71) and the first elected president of the society (1971–73). In addition to his scientific work he had a keen interest in science education establishing the successful Eugene and Ruth Roberts Summer Student Academy at the City of Hope in 1960. This effort enabled thousands of talented high school and college students to experience biomedical research first hand by actually doing it. Eugene remained active, curious and enthusiastic about science until the end. His early research in cancer came together with GABA when he was ask to collaborate on a project dealing with breast cancer metastases to the brain (Neman et al. 2014). Dr. Roberts was an inspiration and mentor to many neuroscientists as well as countless others who knew him from his work. He is survived by his beloved wife and scientific partner of over 40 years, Ruth Roberts (the designer of the ASN logo). The authors declare no conflict of interest. Photograph courtesy of Ruth Roberts and used with permission.
Abstract The ultrastructural features of cholinergic neurons transplanted to the rat hippocampal formation were studied by using a monoclonal antibody to choline acetyltransferase (ChAT). Septal cell suspensions were prepared from E‐18 rat embryos and injected into the hippocampus of host rats that had been previously subjected to a bilateral transection of the fimbria‐fornix. Rats with fimbria‐fornix lesions alone and unoperated rats served as controls and were examined to characterize the native hippocampal cholinergic system. Both unoperated controls and rats with fimbria‐fornix lesions showed a sparse population of intrinsic ChAT‐immunoreactive neurons that were most numerous in the subgranular zone, the hilus fascia dentata, and near the hippocampal fissure. ChAT‐positive terminals from controls formed synapses on dendritic structures that were primarily symmetrical. ChAT‐positive dendrites in controls received synaptic input from nonimmunoreactive axon terminals. In rats with septal transplants, ChAT‐immunoreactive transplant neurons were found that were either bipolar or multipolar. Axons of transplanted neurons were unmyelinated and arose either from the cell body or a primary dendritic process where they gave off numerous collaterals. Terminals from transplant neurons formed synapses with many nonimmunoreactive neurons. In transplant animals, two main targets of ChAT‐immunoreactive terminals were identified: (1) The great majority of synapses were symmetrical junctions with dendritic spines and shafts. (2) A number of terminals were found that appeared to be juxtaposed to nonimmunoreactive axon terminals, possibly forming symmetrical axo‐axonic connections. In contrast, such axo‐axonic contacts were not observed in the controls. It is concluded that transplanted cholinergic neurons may reinnervate the host hippocampus; however, this reinnervation is different from what is seen in the intact hippocampal formation.
Abstract— [ 125 I]Diiodo α‐bungarotoxin ([ 125 I] 2 BuTx) and [ 3 H]quinuclidinylbenzilate ([ 3 H]QNB) binding sites were measured in post‐nuclear membrane fractions prepared from whole brains or brain regions of several species. Species studied included Drosophila melanogaster (fruit fly), Torpedo californiea (electric ray), Carassius auratus (goldfish), Ram pipiens (grass frog), Kana cutesheiana (bullfrog), Rattus norvegicus (rat, Sprague‐Dawley), Mus muscalus (mouse, Swiss random, C58/J, LG/J), Oryctolagus cuniculus (rabbit, New Zealand Whitc), and Bos (cow). Acetyl‐CoA: choline O ‐acetyltransferase (EC 2.3.1.6) levels were also determined in the post nuclear supernatants and correlated with the number of binding sites. All species and regions except Drosophila had 16–150 fold more [ 3 H]QNB binding sites than [ 125 I] 2 BuTx binding sites. Brain regions with the highest levels of [ 125 I] 2 BuTx binding were Drosophila heads (300 fmol/mg), goldfish optic tectum (80fmol/mg), and rat and mouse hippocampus (3040 fmol/mg). The highest levels of [ 3 H]QNB binding were seen in rat and mouse caudate (1.3–1.6 pmol/mg). Lowest levels of [ 3 H]QNB and [ 125 I] 2 BuTx binding were seen in cerebellum. The utility of [ 125 I] 2 BuTx and [ 3 H]QNB binding as quantitative measures of nicotinic and muscarinic acetylcholine receptors in CNS is discussed.
We have constructed transgenic Drosophila melanogaster lines that express green fluorescent protein (GFP) exclusively in the nervous system. Expression is controlled with transcriptional regulatory elements present in the 5′ flanking DNA of the Drosophila Na + ,K + -ATPase β-subunit gene Nervana2 ( Nrv2 ). This regulatory DNA is fused to the yeast transcriptional activator GAL4, which binds specifically to a sequence motif termed the UAS (upstream activating sequence). Drosophila lines carrying Nrv2 -GAL4 transgenes have been genetically recombined with UAS–GFP (S65T) transgenes ( Nrv2- GAL4+UAS–GFP) inserted on the same chromosomes. We observe strong nervous system-specific fluorescence in embryos, larvae, pupae, and adults. The GFP fluorescence is sufficiently bright to allow dynamic imaging of the nervous system at all of these developmental stages directly through the cuticle of live Drosophila . These lines provide an unprecedented view of the nervous system in living animals and will be valuable tools for investigating a number of developmental, physiological, and genetic neurobiological problems.
Abstract Choline acetyltransferase (ChAT), the acetylcholine‐synthesizing enzyme and a definitive marker for cholinergic neurons, was localized immunocytochemically in the motor and somatic sensory regions of rat cerebral cortex with monoclonal antibodies. ChAT‐positive (ChAT+) varicose fibers and terminal‐like structures were distributed in a loose network throughout the cortex. Some immunoreactive cortical fibers were continuous with those in the white matter underlying the cortex, and many of these fibers presumably originated from subcortical cholinergic neurons. ChAT+ fibers appeared to be rather evenly distributed throughout all layers of the motor cortex, but a subtle laminar pattern was evident in the somatic sensory cortex, where lower concentrations of fibers in layer IV contrasted with higher concentrations in layer V. Electron microscopy demonstrated that immunoreaction product was concentrated in synaptic vesicle‐filled profiles and that many of these structures formed synaptic contacts. ChAT+ synapses were present in all cortical layers, and the majority were of the symmetric type, although a few asymmetric ones were also observed. The most common postsynaptic elements were small to medium‐sized dendritic shafts of unidentified origin. In addition, ChAT + terminals formed synaptic contacts with apical and, probably, basilar dendrites of pyramidal neurons, as well as with the somata of ChAT‐negative nonpyramidal neurons. ChAT+ cell bodies were present throughout cortical layers II–VI, but were most concentrated in layers II–III. The somata were small in size, and the majority of ChAT + neurons were bipolar in form, displaying vertically oriented dendrites that often extended across several cortical layers. Electron microscopy confirmed the presence of immunoreaction product within the cytoplasm of small neurons and revealed that they received both symmetric and asymmetric synapses on their somata and proximal dendrites. These observations support an identification of ChAT + cells as nonpyramidal intrinsic neurons and thus indicate that there is an intrinsic source of cholinergic innervation of the rat cerebral cortex, as well as the previously described extrinsic sources.
Abnormal accumulation of Aβ (amyloid β) within AEL (autophagy–endosomal–lysosomal) vesicles is a prominent neuropathological feature of AD (Alzheimer's disease), but the mechanism of accumulation within vesicles is not clear. We express secretory forms of human Aβ 1–40 or Aβ 1–42 in Drosophila neurons and observe preferential localization of Aβ 1–42 within AEL vesicles. In young animals, Aβ 1–42 appears to associate with plasma membrane, whereas Aβ 1–40 does not, suggesting that recycling endocytosis may underlie its routing to AEL vesicles. Aβ 1–40 , in contrast, appears to partially localize in extracellular spaces in whole brain and is preferentially secreted by cultured neurons. As animals become older, AEL vesicles become dysfunctional, enlarge and their turnover appears delayed. Genetic inhibition of AEL function results in decreased Aβ 1–42 accumulation. In samples from older animals, Aβ 1–42 is broadly distributed within neurons, but only the Aβ 1–42 within dysfunctional AEL vesicles appears to be in an amyloid-like state. Moreover, the Aβ 1–42 -containing AEL vesicles share properties with AD-like extracellular plaques. They appear to be able to relocate to extracellular spaces either as a consequence of age-dependent neurodegeneration or a non-neurodegenerative separation from host neurons by plasma membrane infolding. We propose that dysfunctional AEL vesicles may thus be the source of amyloid-like plaque accumulation in Aβ 1–42 -expressing Drosophila with potential relevance for AD.
