Survival and Axonal Outgrowth of the Mauthner Cell Following Spinal Cord Crush Does Not Drive Post-injury Startle Responses.

A pair of Mauthner cells (M-cells) can be found in the hindbrain of most teleost fish, as well as amphibians and lamprey. The axons of these reticulospinal neurons cross the midline and synapse on interneurons and motoneurons as they descend the length of the spinal cord. The M-cell initiates fast C-type startle responses (fast C-starts) in goldfish and zebrafish triggered by abrupt acoustic/vibratory stimuli. Starting about 70 days after whole spinal cord crush, less robust startle responses with longer latencies manifest in adult goldfish, Carassius auratus. The morphological and electrophysiological identifiability of the M-cell provides a unique opportunity to study cellular responses to spinal cord injury and the relation of axonal regrowth to a defined behavior. After spinal cord crush at the spinomedullary junction about one-third of the damaged M-axons of adult goldfish send at least one sprout past the wound site between 56 and 85 days postoperatively. These caudally projecting sprouts follow a more lateral trajectory relative to their position in the fasciculus longitudinalis medialis of control fish. Other sprouts, some from the same axon, follow aberrant pathways that include rostral projections, reversal of direction, midline crossings, neuromas, and projection out the first ventral root. Stimulating M-axons in goldfish that had post-injury startle behavior between 198 and 468 days postoperatively resulted in no or minimal EMG activity in trunk and tail musculature as compared to control fish. Although M-cells can survive for at least 468 day (∼1.3 years) after spinal cord crush, maintain regrowth, and elicit putative trunk EMG responses, the cell does not appear to play a substantive role in the emergence of acoustic/vibratory-triggered responses. We speculate that aberrant pathway choice of this neuron may limit its role in the recovery of behavior and discuss structural and functional properties of alternative candidate neurons that may render them more supportive of post-injury startle behavior.

Invasion of microglia/macrophages and granulocytes into the Mauthner axon myelin sheath following spinal cord injury of the adult goldfish, Carassius auratus.

Rapid activation of resident glia occurs after spinal cord injury. Somewhat later, innate and adaptive immune responses occur with the invasion of peripheral immune cells into the wound site. The activation of resident and peripheral immune cells has been postulated to play harmful as well as beneficial roles in the regenerative process. Mauthner cells, large identifiable neurons located in the hindbrain of most fish and amphibians, provided the opportunity to study the morphological relationship between reactive cells and Mauthner axons (M-axons) severed by spinal cord crush or by selective axotomy. After crossing in the hindbrain, the M-axons of adult goldfish, Carassius auratus, extend the length of the spinal cord. Following injury, the M-axon undergoes retrograde degeneration within its myelin sheath creating an axon-free zone (proximal dieback zone). Reactive cells invade the wound site, enter the axon-free dieback zone and are observed in the vicinity of the retracted M-axon tip as early as 3 hr postinjury. Transmission electron microscopy allowed the detection of microglia/macrophages and granulocytes, some of which appear to be neutrophil-like, at each of these locations. We believe that this is the first report of the invasion of such cells within the myelin sheath of an identifiable axon in the vertebrate central nervous system (CNS). We speculate that microglia/macrophages and granulocytes that are attracted within a few hours to the damaged M-axon are part of an inflammatory response that allows phagocytosis of debris and plays a role in the regenerative process. Our results provide the baseline from which to utilize immunohistochemical and genetic approaches to elucidate the role of non-neuronal cells in the regenerative process of a single axon in the vertebrate CNS.

Hypoxia Has a Lasting Effect on Fast-Startle Behavior of the Tropical Fish Haemulon plumieri.

Anthropogenic activities and climate change have resulted in an increase of hypoxic conditions in nearshore ecosystems worldwide. Depending on the persistence of a hypoxic event, the survival of aquatic animals can be compromised. Temperate fish exposed to hypoxia display a reduction in the probability of eliciting startle responses thought to be important for escape from predation. Here we examine the effect of hypoxia on the probability of eliciting fast-startle responses (fast-starts) of a tropical fish, the white grunt (Haemulon plumieri), and whether hypoxia has a prolonged impact on behavior once the fish are returned to normoxic conditions. White grunts collected from the San Juan Bay Estuary in Puerto Rico were exposed to an oxygen concentration of 2.5 mg L-1 (40% dissolved oxygen). We found a significant reduction in auditory-evoked fast-starts that lasted for at least 24 hours after fish were returned to normoxic conditions. Accessibility to the neuronal networks that underlie startle responses was an important motivator for this study. Mauthner cells are identifiable neurons found in most fish and amphibians, and these cells are known to initiate fast-starts in teleost fishes. The assumption that most of the short-latency responses in this study are Mauthner cell initiated provided the impetus to characterize the white grunt Mauthner cell. The identification of the cell provides a first step in understanding how low oxygen levels may impact a single cell and its circuit and the behavior it initiates.

Mary Jane Hogue (1883-1962): A pioneer in human brain tissue culture.

The ability to maintain human brain explants in tissue culture was a critical step in the use of these cells for the study of central nervous system disorders. Ross G. Harrison (1870-1959) was the first to successfully maintain frog medullary tissue in culture in 1907, but it took another 38 years before successful culture of human brain tissue was accomplished. One of the pioneers in this achievement was Mary Jane Hogue (1883-1962). Hogue was born into a Quaker family in 1883 in West Chester, Pennsylvania, and received her undergraduate degree from Goucher College in Baltimore, Maryland. Research with the developmental biologist Theodor Boveri (1862-1915) in Würzburg, Germany, resulted in her Ph.D. (1909). Hogue transitioned from studying protozoa to the culture of human brain tissue in the 1940s and 1950s, when she was one of the first to culture cells from human fetal, infant, and adult brain explants. We review Hogue's pioneering contributions to the study of human brain cells in culture, her putative identification of progenitor neuroblast and/or glioblast cells, and her use of the cultures to study the cytopathogenic effects of poliovirus. We also put Hogue's work in perspective by discussing how other women pioneers in tissue culture influenced Hogue and her research.

The Marine Biological Laboratory (Woods Hole) and the scientific advancement of women in the early 20th century: the example of Mary Jane Hogue (1883-1962).

The Marine Biological Laboratory (MBL) in Woods Hole, MA provided opportunities for women to conduct research in the late 19th and early 20th century at a time when many barriers existed to their pursuit of a scientific career. One woman who benefited from the welcoming environment at the MBL was Mary Jane Hogue. Her remarkable career as an experimental biologist spanned over 55 years. Hogue was born into a Quaker family in 1883 and received her undergraduate degree from Goucher College. She went to Germany to obtain an advanced degree, and her research at the University of Würzburg with Theodor Boveri resulted in her Ph.D. (1909). Although her research interests included experimental embryology, and the use of tissue culture to study a variety of cell types, she is considered foremost a protozoologist. Her extraordinary demonstration of chromidia (multiple fission) in the life history of a new species of Flabellula associated with diseased oyster beds is as important as it is ignored. We discuss Hogue's career path and her science to highlight the importance of an informal network of teachers, research advisors, and other women scientists at the MBL all of whom contributed to her success as a woman scientist.

On the training of future neuroscientists: insights from the Grass laboratory.

The understanding of nature is a continuous process that requires the transference of current knowledge to future generations. In this NeuroView, we address the critical issue of training of future scientists, an essential aspect of scientific progress. As an example of the impact training programs can have on shaping future scientists, we focus on the experience of the Grass Laboratory, which provides early career investigators the opportunity to embark on independent research experiences. This uniquely designed program has contributed enormously to fostering the development of neuroscientists in the past 60 years and has left a recognizable mark on 20(th) and 21(st) century neuroscience research.

How the early voltage clamp studies of José del Castillo inform "modern" neuroscience.

The description of ionic currents that flow across the membrane of the squid giant axon during an action potential sparked an interest in determining whether there were similar currents in vertebrates. The preparation of choice was the node of Ranvier in single myelinated fibers in frog. José del Castillo spent 3 years on the United States mainland from 1956 to 1959. During that time, he collaborated with Jerome Y. Lettvin and John W. Moore. I discuss how these individuals met one another and some of their scientific discoveries using the voltage clamp to study squid giant axons and frog nodes. Much of this work was conducted at the Marine Biological Laboratory in Woods Hole, MA, and I attempt to convey a sense of the unique scientific "melting pot" that existed at the Marine Biological Laboratory and the broader effect that del Castillo had on "modern" neuroscience.

Regeneration in the era of functional genomics and gene network analysis.

What gives an organism the ability to regrow tissues and to recover function where another organism fails is the central problem of regenerative biology. The challenge is to describe the mechanisms of regeneration at the molecular level, delivering detailed insights into the many components that are cross-regulated. In other words, a broad, yet deep dissection of the system-wide network of molecular interactions is needed. Functional genomics has been used to elucidate gene regulatory networks (GRNs) in developing tissues, which, like regeneration, are complex systems. Therefore, we reason that the GRN approach, aided by next generation technologies, can also be applied to study the molecular mechanisms underlying the complex functions of regeneration. We ask what characteristics a model system must have to support a GRN analysis. Our discussion focuses on regeneration in the central nervous system, where loss of function has particularly devastating consequences for an organism. We examine a cohort of cells conserved across all vertebrates, the reticulospinal (RS) neurons, which lend themselves well to experimental manipulations. In the lamprey, a jawless vertebrate, there are giant RS neurons whose large size and ability to regenerate make them particularly suited for a GRN analysis. Adding to their value, a distinct subset of lamprey RS neurons reproducibly fail to regenerate, presenting an opportunity for side-by-side comparison of gene networks that promote or inhibit regeneration. Thus, determining the GRN for regeneration in RS neurons will provide a mechanistic understanding of the fundamental cues that lead to success or failure to regenerate.

Axon cap morphology of the sea robin (Prionotus carolinus): Mauthner cell is correlated with the presence of "signature" field potentials and a C-type startle response.

Studies on the Mauthner cell (M-cell) of goldfish, Carassius auratus, have facilitated our understanding of how sensory information is integrated in the hindbrain to initiate C-type fast startle responses (C-starts). The goldfish M-cell initial segment/axon hillock is surrounded by a composite axon cap consisting of a central core and a peripheral zone covered by a glial cell layer. The high resistivity of the axon cap results in "signature" field potentials recorded on activation of the M-cell, allowing unequivocal physiological identification of the M-cell and of its feedback and reciprocal inhibitory networks that are crucial in ensuring that only one M-cell is active and that it fires only once. Phylogenetic mapping of axon cap morphology to muscle activity patterns and behavior predicts that teleost fishes that have a composite axon cap, like that of the goldfish, will perform C-start behavior with primarily unilateral muscle activity. We have chosen to study these predictions in the northern sea robin, Prionotus carolinus, a percomorph fish. Although sea robins have a very different phylogenetic position, body form, and habitat compared with the goldfish, they display the correlation of axon cap morphology to physiology and C-start behavior. Differences in response parameters suggest some evolutionary trade-offs in sea robin C-start behavior compared with that of the goldfish, but the correlations in morphology, physiology, and behavior are common features of both otophysan and nonotophysan teleosts. The M-cell will continue to provide an unprecedented opportunity to study the evolution of a neural circuit in the context of behavior.

Evolution of the Mauthner axon cap.

Studies of vertebrate brain evolution have focused primarily on patterns of gene expression or changes in size and organization of major brain regions. The Mauthner cell, an important reticulospinal neuron that functions in the startle response of many species, provides an opportunity for evolutionary comparisons at the cellular level. Despite broad interspecific similarities in Mauthner cell morphology, the motor patterns and startle behaviors it initiates vary markedly. Response diversity has been hypothesized to result, in part, from differences in the structure and function of the Mauthner cell-associated axon cap. We used light microscopy techniques to compare axon cap morphology across a wide range of species, including all four extant basal actinopterygian orders, representatives of a variety of teleost lineages and lungfishes, and we combined our data with published descriptions of axon cap structure. The 'composite' axon cap, observed in teleosts, is an organized conglomeration of glia and fibers of inhibitory and excitatory interneurons. Lungfish, amphibian tadpoles and several basal actinopterygian fishes have 'simple' axon caps that appear to lack glia and include few fibers. Several other basal actinopterygian fishes have 'simple-dense' caps that include greater numbers of fibers than simple caps, but lack the additional elements and organization of composite caps. Phylogenetic mapping shows that through evolution there are discrete transitions in axon cap morphology occurring at the base of gnathostomes, within basal actinopterygians, and at the base of the teleost radiation. Comparing axon cap evolution to the evolution of startle behavior and motor pattern provides insight into the relationship between Mauthner cell-associated structures and their functions in behavior.

Reticulospinal neurons in anamniotic vertebrates: a celebration of Alberto Stefanelli's contributions to comparative neuroscience.

Over the past 76 years Alberto Stefanelli has successfully used a comparative approach to study the nervous system. His main research focus during that time has been on identifiable reticulospinal neurons including Müller and Mauthner neurons found in anamniotic vertebrates. Born in Venice, Italy in 1908, Professor Stefanelli pursued most of his academic career at the University of Rome, where he retired as Chair of Comparative Anatomy in 1978. His seminal work on the constancy in number and position of giant identifiable reticulospinal neurons in the brains of larval and adult lampreys, and his assertion that only a subset of these neurons were Müller cells, provided the framework in which subsequent authors have refined our understanding of the cellular anatomy, axonal projections, physiology, and function of Müller cells in the control of movement. Stefanelli has also provided the most comprehensive study to date of the Mauthner cell and its axon cap. His description of the differences in axon cap structure among many fishes and amphibians and his use of the "morpho-ecological" approach to determine Mauthner cell function has provided the basis for future studies on the neuronal basis of behavior and its evolution. As Professor Stefanelli approaches his 100th birthday, we celebrate his scientific contributions to comparative neuroscience with a biographical sketch of his life, an overview of his scientific accomplishments, and our view of how his comparative studies continue to contribute to our understanding of the nervous system.

Correlation of C-start behaviors with neural activity recorded from the hindbrain in free-swimming goldfish (Carassius auratus).

Startle behaviors in teleost fishes are well suited for investigations of mechanisms of sensorimotor integration because the behavior is quantifiable and much of the underlying circuitry has been identified. The teleost C-start is triggered by an action potential in one of the two Mauthner (M) cells. To correlate C-start behavior with electrophysiology, extracellular recordings were obtained from the surface of the medulla oblongata in the hindbrain, close to the M-axons, in freely swimming goldfish monitored using high-speed video. The recordings included action potentials generated by the two M-axons, as well as neighboring axons in the dorsal medial longitudinal fasciculus. Axonal backfills indicated that the latter originate from identifiable reticulospinal somata in rhombomeres 2-8 and local interneurons. Diverse auditory and visual stimuli evoked behaviors with kinematics characteristic of the C-start, and the amplitude of the first component of the hindbrain field potential correlated with the C-start direction. The onset of the field potential preceded that of the simultaneously recorded trunk EMG and movement initiation by 1.08+/-0.04 and 8.13+/-0.17 ms, respectively. A subsequent longer latency field potential was predictive of a counterturn. These results indicate that characteristic features of the C-start can be extracted from the neural activity of the M-cell and a population of other reticulospinal neurons in free-swimming goldfish.

The effects of head and tail stimulation on the withdrawal startle response of the rope fish (Erpetoichthys calabaricus).

While most actinopterygian fishes perform C-start or S-start behaviors as their primary startle responses, many elongate species instead use a withdrawal movement. Studies of withdrawal have focused on the response to head-directed or nonspecific stimuli. During withdrawal, the animal moves its head back from the stimulus, often resulting in several tight bends in the body. In contrast to C-start or S-start behaviors, withdrawal to a head stimulus generally does not involve a subsequent propulsive stage of movement. We examined intraspecific diversity in withdrawal behavior and muscle activity patterns of the rope fish, Erpetoichthys calabaricus, in response to stimulation of the head and the tail. In addition, we describe the anatomy of the Mauthner cells and their axon caps, structures that are generally absent in species with a withdrawal startle. We recorded high-speed video (250 Hz) and electromyograms (EMGs) from 12 electrodes in the axial muscle during the behavioral response. We used Bodian silver staining techniques to visualize Mauthner cell and axon cap morphology. We found that E. calabaricus responds with a withdrawal to both head and tail stimulation. Tail stimulation elicits a stronger kinematic and muscle activity response than head stimulation. While withdrawal movement generally constitutes the entire response to head stimuli, withdrawal was followed by propulsive movements when the tail was stimulated, suggesting that withdrawal can both act alone and serve as the first stage of a propulsive startle. Unexpectedly, bilaterality of muscle activity was variable for responses to both head and tail stimuli. In addition, we were surprised to find that E. calabaricus has a distinct axon cap associated with its Mauthner cell. These data suggest that the withdrawal response is a more diverse functional system than has previously been believed.