Boards, Advisory Committees, Professional Organizations
Co-Director, SURMAR/ASIMAR (The Ocean Foundation) (2008 - Present)
External Advisory Board member, Puerto Rico Center for Enviornmental Neuroscience (PRCEN), Univ. Puerto Rico (2012 - Present)
Board member, Western Flyer Foundation (2016 - Present)
Postdoctoral, University of Pennsylvania, Biology, Physiology (1979)
PhD, Washington University School of Medicine, Yale University School of Medicine, Physiology and Biophysics (1978)
BSE, Princeton University, Electrical Engineering (1972)
Community and International Work
Squids4Kids, Pacific Grove, CA
Ocean health and marine ecology
NOAA Southwest Fisheries Sciene Center, Monterey Bay National Marine Sancturary Foundation
K-12, science festivals
Opportunities for Student Involvement
Sustainable Utilization and Research of Mar de Cortes (SURMAR), Santa Rosalia, BCS, Mexico
Marine ecology and resource develeopment
Intsiuto Tecnologico Superior de Mulege, The Ocean Foundation
Mexican fishing communities and undergraduate students
Opportunities for Student Involvement
Current Research and Scholarly Interests
My group was the first (and only) to deploy pop-up satellite tags and video packages (National Geographic Crittercam) on large Humboldt squid to record their second-to-second movements and color-changing behaviors. This work showed that this active predator spends a great deal of its time at depths of 300 m or more where the oxygen concentration is extremely low – less than 10% of that at the surface. This ‘oxygen minim zone’ (OMZ) is found throughout the southern half of the Gulf of California and much of the eastern Pacific Ocean, including Monterey Bay. The OMZ has been moving closer to the sea surface over the last few decades, and this aspect of marine climate change is expected to have major ecological consequences as ocean’s oxygenated surface zone becomes increasingly vertically compressed.
Our work in the Gulf of California has recently focused on the relationship between the size of Humboldt squid and environmental variation, particularly temperature at depth. Since an unusual El Niño event in 2009-10, the temperature at depths of up to 100 m has been increasing, and squid have responded by attaining maturity at a vastly smaller size and younger age than they had before 2009. Small size at maturity is normally a characteristic phenotype of this species in the tropical eastern Pacific, and the change in the squid’s life history in the Sea of Cortez is consistent with the decreasing productivity and increasing temperatures observed over the last 6 years. Humboldt squid are telling us that the Gulf of California may be changing from a seasonally highly productive, upwelling-driven system to a low productivity tropical system.
Current laboratory work on squid chromatophores uses methods of electrophysiology, cell and molecular biology and electron microscopy, through collaborations with Univ. Puerto Rico, Univ. North Carolina Chapel Hill and Univ. Kansas. A major hypothesis guiding the work is that a “horizontal” pathway for communication between chromatophores exists in the plane of the skin, and that this network can mediate chromogenic behaviors in the absence of descending motor control by the central nervous system. We use a comparative approach to take advantage of natural differences in behavioral capabilities of Humboldt squid (Dosidicus gigas) and CA market squid (Doryteuthis opalescens) that inhabit environments with extremely different visual features. Market squid are a coastal species that use spatial patterning of chromatophore displays to provide camouflage in order to match benthic features like seaweed and rocks. Humboldt squid are an open ocean species that primarily generate temporal patterning and use spatially global flashing in intra-specific signaling. We hypothesize that these striking behavioral differences will be reflected in structural and functional elements of the peripheral control pathway.
Another project examines the role of the giant axon system in controlling escape responses in both Dosidicus and Doryteuthis, with a focus on sensitivity of the system to temperature and hypoxia. Both of these environmental variables are relevant to these species in the ocean. Methods used include electrophysiology, anatomy and particle image velocimetry.
Laboratory work is carried out both at Hopkins Marine Station and at our lab facility in Santa Rosalia, BCS, Mexico.
Natural Chromogenic Behaviors of Squid in Oceanic Waters, Stanford Univesity (6/1/2014 - 5/31/2017)
1. Record natural behaviors in free-swimming Humboldt squid (Dosidicus gigas) in the water column under natural lighting conditions using low-light video packages to characterize dynamic chromogenic displays that are related to intra-specific signaling and crypsis.
2. Develop improved low-light, free-floating video packages to image behavior of marine organisms under natural lighting conditions at midwater depths, including chromogenic behaviors of squid, and to make these packages available to other researchers.
3. Compare chromogenic behaviors and underlying structural and functional features of the chromatophore system in squid species that inhabit distinct environments with different visual features -- open ocean for Dosidicus gigas (family Ommastrephidae) versus coastal shallows for Doryteuthis opalescens, the California market squid (family Loliginidae).
Hopkins Marirne Station and Santa Rosalia, Baja California Sur, Mexico
- Eric Berkenpas, Lead Engineer, National Geographic Remote Imaging
- Mike Shepard, Engineer, National Geographic Remote Imaging
Collaborative Research: Structural and Functional Connectivity of Squid Chromatophores, Stanford Univesity (7/1/2016 - 6/30/2019)
Squid and other cephalopods have the ability to change skin color using muscular chromatophore organs that are under direct neural control. All work on the cellular mechanisms of chromatophore control in squid has focused on three species in the family Loliginidae that inhabit coastal environments rich in benthic features like seaweed, rocks and coral. Skin-color changes in these species are associated with camouflage as well as intra-specific signaling and deimatic displays. The open ocean presents a radically different environment that is also inhabited by many squids, primarily of the family Ommastrephidae, one that includes the Humboldt squid (Dosidicus gigas). There is little light in the oceanic water column at depths inhabited by these squid during daytime, and static visual features are non-existent. Novel color-change behaviors in Dosidicus include repetitive whole-body “flashing,” used for intra-specific signaling, and chaotic-like “flickering” that may underlie crypsis in the open ocean. Although these dynamic behaviors contrast with the generally more static patterns in loliginids, squids of both families employ temporal and spatial patterning to varying degrees. It is therefore likely that basic mechanisms for controlling the chromatophore network are shared by most, if not all, squids. Descending “vertical” control from the brain to the chromatophore musculature is well established in loliginids and may account for most chromogenic behaviors in those species, but behaviors in ommastrephids like flickering may be more influenced by processes within the skin itself that permit excitation to spread from one chromatophore to another without directly involving the nervous system. This hypothetical pathway would define a “horizontal” or distributed control system in the periphery that would permit autonomous behavior within the chromatophore network. Horizontal control is relevant to the vascular bed, gut and coupled neural micro-circuits in vertebrates, and results from this project will thus influence this broader field. From a wider perspective, results of this project will be relevant to interactions of distributed (horizontal) and top-down (vertical) control mechanisms, an inherent feature of complex systems to generate non-predictable, emergent phenomena. This concept is of fundamental interest to a broad sector of society, ranging from engineering to economics to politics.
An integrated approach will be followed to test the hypothesis that control of the chromatophore network in squid involves peripheral mechanisms that are distinct from the neuronal motor-control pathway that descends from the brain. Spontaneous chromatophore activity that is independent of canonical neural control will be isolated by experimental manipulations in loliginid squid (Doryteuthis opalescens), including chronic denervation and pharmacological block of neuronal activity with tetrodotoxin. In addition, a comparative approach will take advantage of an ommastrephid species, Dosidicus gigas, in which spontaneous, tetrodotoxin-resistant chromatophore activity is extremely prominent. Relevant methods involve cellular electrophysiology, molecular transcriptomics, immunohistochemistry with confocal microscopy and high-resolution electron microscopy. Specific aims are: 1) identify molecular and physiological properties of relevant ion channels and receptors that control excitability in the radial muscle fibers that operate individual chromatophore organs in Doryteuthis; 2) define structural, molecular and physiological features of coupling mechanisms between muscle fibers of neighboring chromatophores that define an excitatory transmission pathway within the skin; 3) elucidate the inhibitory role in controlling spontaneous chromatophore activity played by serotonin; 4) carry out parallel experiments in Dosidicus, a member of a family of ecologically important squid in which cellular studies of chromatophores have never been carried out.
Hopkins Marirne Station and Santa Rosalia, Baja California Sur, Mexico
- Josh Rosenthal, Professor, University of Puerto Rico
- Bill Kier, Professsor, University of North Carolina, Chapel Hill
Variations in water column properties of the Sea of Cortez in relation to ecosystem and climate dynamics, Stanford University and SURMAR (The Ocean Foundation) (12/1/2015 - November 30, 2018)
Climate change is a significant threat to the ecological balance in the Gulf of California and long-term measurement of both oceanographic and biological/ecological features are necessary to understand the impacts of climate change. These effects can be exerted seasonally (the Gulf has a huge temperature variation), over the course of a year or two by a strong El Niño, or over decades by global warming and the associated phenomena of ocean acidification and oxygen minimum zone expansion. The latter change probably involves agricultural runoff and potentially sewage discharge. Although remote sensing of ocean surface properties is an excellent and cost-effective way of tracking important aspects of climate change, it cannot discern changes that are occurring hundreds of meters below the surface, and this midwater environment is critically important, either directly or indirectly, to almost all pelagic organisms, including Humboldt squid. Systematic measurements of water-column properties in the field are essential; there is simply no substitute.
This project is designed to continue a monitoring program of water-column prperties in the Gulf of Califronia that began in 2010. Measurements of temperature, salinity, oxygen and cholorophyl concentrations are made to depths of 600 m using a standard profiling instrument from the vessel National Geographic Seabird as part of ongoing operations by Lindblad Expeditions. To our knowledge, there is no other systematic assessment of water-column properties being carried out in the Gulf of California in the area under consideration.
Hopkins Marirne Station and Baja California Sur, Mexico
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- Prolonged decline of jumbo squid (Dosidicus gigas) landings in the Gulf of California is associated with chronically low wind stress and decreased chlorophyll a after El Nino 2009-2010 FISHERIES RESEARCH 2016; 173: 128-138
Chromogenic behaviors of the Humboldt squid (Dosidicus gigas) studied in situ with an animal-borne video package.
journal of experimental biology
2015; 218: 265-275
Dosidicus gigas (Humboldt or jumbo flying squid) is an economically and ecologically influential species, yet little is known about its natural behaviors because of difficulties in studying this active predator in its oceanic environment. By using an animal-borne video package, National Geographic's Crittercam, we were able to observe natural behaviors in free-swimming D. gigas in the Gulf of California with a focus on color-generating (chromogenic) behaviors. We documented two dynamic displays without artificial lighting at depths of up to 70 m. One dynamic pattern, termed 'flashing' is characterized by a global oscillation (2-4 Hz) of body color between white and red. Flashing was almost always observed when other squid were visible in the video frame, and this behavior presumably represents intraspecific signaling. Amplitude and frequency of flashing can be modulated, and the phase relationship with another squid can also be rapidly altered. Another dynamic display termed 'flickering' was observed whenever flashing was not occurring. This behavior is characterized by irregular wave-like activity in neighboring patches of chromatophores, and the resulting patterns mimic reflections of down-welled light in the water column, suggesting that this behavior may provide a dynamic type of camouflage. Rapid and global pauses in flickering, often before a flashing episode, indicate that flickering is under inhibitory neural control. Although flashing and flickering have not been described in other squid, functional similarities are evident with other species.
View details for DOI 10.1242/jeb.114157
View details for PubMedID 25609785
Evolutionary history of a complex adaptation: Tetrodotoxin resistance in salamanders
2015; 69 (1): 232-244
Understanding the processes that generate novel adaptive phenotypes is central to evolutionary biology. We used comparative analyses to reveal the history of tetrodotoxin (TTX) resistance in TTX-bearing salamanders. Resistance to TTX is a critical component of the ability to use TTX defensively but the origin of the TTX-bearing phenotype is unclear. Skeletal muscle of TTX-bearing salamanders (modern newts, family: Salamandridae) is unaffected by TTX at doses far in excess of those that block action potentials in muscle and nerve of other vertebrates. Skeletal muscle of non-TTX-bearing salamandrids is also resistant to TTX but at lower levels. Skeletal muscle TTX resistance in the Salamandridae results from the expression of TTX-resistant variants of the voltage-gated sodium channel NaV 1.4 (SCN4a). We identified four substitutions in the coding region of salSCN4a that are likely responsible for the TTX resistance measured in TTX-bearing salamanders and variation at one of these sites likely explains variation in TTX resistance among other lineages. Our results suggest that exaptation has played a role in the evolution of the TTX-bearing phenotype and provide empirical evidence that complex physiological adaptations can arise through the accumulation of beneficial mutations in the coding region of conserved proteins.
View details for DOI 10.1111/evo.12552
View details for Web of Science ID 000347462800018
View details for PubMedID 25346116
- Behavioral ecology of jumbo squid (Dosidicus gigas) in relation to oxygen minimum zones DEEP-SEA RESEARCH PART II-TOPICAL STUDIES IN OCEANOGRAPHY 2013; 95: 197-208
Extreme plasticity in life-history strategy allows a migratory predator (jumbo squid) to cope with a changing climate
GLOBAL CHANGE BIOLOGY
2013; 19 (7): 2089-2103
Dosidicus gigas (jumbo or Humboldt squid) is a semelparous, major predator of the eastern Pacific that is ecologically and commercially important. In the Gulf of California, these animals mature at large size (>55 cm mantle length) in 1-1.5 years and have supported a major commercial fishery in the Guaymas Basin during the last 20 years. An El Niño event in 2009-2010, was accompanied by a collapse of this fishery, and squid in the region showed major changes in the distribution and life-history strategy. Large squid abandoned seasonal coastal-shelf habitats in 2010 and instead were found in the Salsipuedes Basin to the north, an area buffered from the effects of El Niño by tidal upwelling and a well-mixed water column. The commercial fishery also relocated to this region. Although large squid were not found in the Guaymas Basin from 2010 to 2012, small squid were abundant and matured at an unusually small mantle-length (<30 cm) and young age (approximately 6 months). Juvenile squid thus appeared to respond to El Niño with an alternative life-history trajectory in which gigantism and high fecundity in normally productive coastal-shelf habitats were traded for accelerated reproduction at small size in an offshore environment. Both small and large mature squid, were present in the Salsipuedes Basin during 2011, indicating that both life- history strategies can coexist. Hydro-acoustic data, reveal that squid biomass in this study area nearly doubled between 2010 and 2011, primarily due to a large increase in small squid that were not susceptible to the fishery. Such a climate-driven switch in size-at-maturity may allow D. gigas to rapidly adapt to and cope with El Niño. This ability is likely to be an important factor in conjunction with longerterm climate-change and the potential ecological impacts of this invasive predator on marine ecosystems.
View details for DOI 10.1111/gcb.12198
View details for Web of Science ID 000319963500010
View details for PubMedID 23505049
Oceanographic and biological effects of shoaling of the oxygen minimum zone.
Annual review of marine science
2013; 5: 393-420
Long-term declines in oxygen concentrations are evident throughout much of the ocean interior and are particularly acute in midwater oxygen minimum zones (OMZs). These regions are defined by extremely low oxygen concentrations (<20-45 μmol kg(-1)), cover wide expanses of the ocean, and are associated with productive oceanic and coastal regions. OMZs have expanded over the past 50 years, and this expansion is predicted to continue as the climate warms worldwide. Shoaling of the upper boundaries of the OMZs accompanies OMZ expansion, and decreased oxygen at shallower depths can affect all marine organisms through multiple direct and indirect mechanisms. Effects include altered microbial processes that produce and consume key nutrients and gases, changes in predator-prey dynamics, and shifts in the abundance and accessibility of commercially fished species. Although many species will be negatively affected by these effects, others may expand their range or exploit new niches. OMZ shoaling is thus likely to have major and far-reaching consequences.
View details for DOI 10.1146/annurev-marine-120710-100849
View details for PubMedID 22809177
Locomotion and behavior of Humboldt squid, Dosidicus gigas, in relation to natural hypoxia in the Gulf of California, Mexico
JOURNAL OF EXPERIMENTAL BIOLOGY
2012; 215 (18): 3175-3190
We studied the locomotion and behavior of Dosidicus gigas using pop-up archival transmitting (PAT) tags to record environmental parameters (depth, temperature and light) and an animal-borne video package (AVP) to log these parameters plus acceleration along three axes and record forward-directed video under natural lighting. A basic cycle of locomotor behavior in D. gigas involves an active climb of a few meters followed by a passive (with respect to jetting) downward glide carried out in a fins-first direction. Temporal summation of such climb-and-glide events underlies a rich assortment of vertical movements that can reach vertical velocities of 3 m s(-1). In contrast to such rapid movements, D. gigas spends more than 80% of total time gliding at a vertical velocity of essentially zero (53% at 0±0.05 m s(-1)) or sinking very slowly (28% at -0.05 to -0.15 m s(-1)). The vertical distribution of squid was compared with physical features of the local water column (temperature, oxygen and light). Oxygen concentrations of ≤20 μmol kg(-1), characteristic of the midwater oxygen minimum zone (OMZ), can influence the daytime depth of squid, but this depends on location and season, and squid can 'decouple' from this environmental feature. Light is also an important factor in determining daytime depth, and temperature can limit nighttime depth. Vertical velocities were compared over specific depth ranges characterized by large differences in dissolved oxygen. Velocities were generally reduced under OMZ conditions, with faster jetting being most strongly affected. These data are discussed in terms of increased efficiency of climb-and-glide swimming and the potential for foraging at hypoxic depths.
View details for DOI 10.1242/jeb.072538
View details for Web of Science ID 000308041400011
View details for PubMedID 22915711
- Coordinated nocturnal behavior of foraging jumbo squid Dosidicus gigas MARINE ECOLOGY PROGRESS SERIES 2012; 455: 211-228
Diversity of conotoxin types from Conus californicus reflects a diversity of prey types and a novel evolutionary history
2011; 57 (2): 311-322
Most species within the genus Conus are considered to be specialists in their consumption of prey, typically feeding on molluscs, vermiform invertebrates or fish, and employ peptide toxins to immobilize prey. Conus californicus Hinds 1844 atypically utilizes a wide range of food sources from all three groups. Using DNA- and protein-based methods, we analyzed the molecular diversity of C. californicus toxins and detected a correspondingly large number of conotoxin types. We identified cDNAs corresponding to seven known cysteine-frameworks containing over 40 individual inferred peptides. Additionally, we found a new framework (22) with six predicted peptide examples, along with two forms of a new peptide type of unusual length. Analysis of leader sequences allowed assignment to known superfamilies in only half of the cases, and several of these showed a framework that was not in congruence with the identified superfamily. Mass spectrometric examination of chromatographic fractions from whole venom served to identify peptides corresponding to a number of cDNAs, in several cases differing in their degree of posttranslational modification. This suggests differential or incomplete biochemical processing of these peptides. In general, it is difficult to fit conotoxins from C. californicus into established toxin classification schemes. We hypothesize that the novel structural modifications of individual peptides and their encoding genes reflect evolutionary adaptation to prey species of an unusually wide range as well as the large phylogenetic distance between C. californicus and Indo-Pacific species.
View details for DOI 10.1016/j.toxicon.2010.12.008
View details for Web of Science ID 000287629400015
View details for PubMedID 21172372
A diverse family of novel peptide toxins from an unusual cone snail, Conus californicus
JOURNAL OF EXPERIMENTAL BIOLOGY
2011; 214 (1): 147-161
Diversity among Conus toxins mirrors the high species diversity in the Indo-Pacific region, and evolution of both is thought to stem from feeding-niche specialization derived from intra-generic competition. This study focuses on Conus californicus, a phylogenetic outlier endemic to the temperate northeast Pacific. Essentially free of congeneric competitors, it preys on a wider variety of organisms than any other cone snail. Using molecular cloning of cDNAs and mass spectrometry, we examined peptides isolated from venom ducts to elucidate the sequences and post-translational modifications of two eight-cysteine toxins (cal12a and cal12b of type 12 framework) that block voltage-gated Na(+) channels. Based on homology of leader sequence and mode of action, these toxins are related to the O-superfamily, but differ significantly from other members of that group. Six of the eight cysteine residues constitute the canonical framework of O-members, but two additional cysteine residues in the N-terminal region define an O+2 classification within the O-superfamily. Fifteen putative variants of Cal12.1 toxins have been identified by mRNAs that differ primarily in two short hypervariable regions and have been grouped into three subtypes (Cal12.1.1-3). This unique modular variation has not been described for other Conus toxins and suggests recombination as a diversity-generating mechanism. We propose that these toxin isoforms show specificity for similar molecular targets (Na(+) channels) in the many species preyed on by C. californicus and that individualistic utilization of specific toxin isoforms may involve control of gene expression.
View details for DOI 10.1242/jeb.046086
View details for Web of Science ID 000285090000024
View details for PubMedID 21147978
Two toxins from Conus striatus that individually induce tetanic paralysis
2006; 45 (47): 14212-14222
We describe structural properties and biological activities of two related O-glycosylated peptide toxins isolated from injected (milked) venom of Conus striatus, a piscivorous snail that captures prey by injecting a venom that induces a violent, spastic paralysis. One 30 amino acid toxin is identified as kappaA-SIVA (termed s4a here), and another 37 amino acid toxin, s4b, corresponds to a putative peptide encoded by a previously reported cDNA. We confirm the amino acid sequences and carry out structural analyses of both mature toxins using multiple mass spectrometric techniques. These include electrospray ionization ion-trap mass spectrometry and nanoelectrospray techniques for small volume samples, as well as matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis as a complementary method to assist in the determination of posttranslational modifications, including O-linked glycosylation. Physiological experiments indicate that both s4a and s4b induce intense repetitive firing of the frog neuromuscular junction, leading to a tetanic contracture in muscle fiber. These effects apparently involve modification of voltage-gated sodium channels in motor axons. Notably, application of either s4a or s4b alone mimics the biological effects of the whole injected venom on fish prey.
View details for DOI 10.1021/bi061485s
View details for Web of Science ID 000242179100029
View details for PubMedID 17115716
Intraspecific variation of venom injected by fish-hunting Conus snails
JOURNAL OF EXPERIMENTAL BIOLOGY
2005; 208 (15): 2873-2883
Venom peptides from two species of fish-hunting cone snails (Conus striatus and Conus catus) were characterized using microbore liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and electrospray ionization-ion trap-mass spectrometry. Both crude venom isolated from the venom duct and injected venom obtained by milking were studied. Based on analysis of injected venom samples from individual snails, significant intraspecific variation (i.e. between individuals) in the peptide complement is observed. The mixture of peptides in injected venom is simpler than that in the crude duct venom from the same snail, and the composition of crude venom is more consistent from snail to snail. While there is animal-to-animal variation in the peptides present in the injected venom, the composition of any individual's injected venom remains relatively constant over time in captivity. Most of the Conus striatus individuals tested injected predominantly a combination of two neuroexcitatory peptides (s4a and s4b), while a few individuals had unique injected-venom profiles consisting of a combination of peptides, including several previously characterized from the venom duct of this species. Seven novel peptides were also putatively identified based on matches of their empirically derived masses to those predicted by published cDNA sequences. Profiling injected venom of Conus catus individuals using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry demonstrates that intraspecific variation in the mixture of peptides extends to other species of piscivorous cone snails. The results of this study imply that novel regulatory mechanisms exist to select specific venom peptides for injection into prey.
View details for DOI 10.1242/jeb.01713
View details for Web of Science ID 000231575800016
View details for PubMedID 16043592
Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of Na(v)1.7 sodium channels in primary afferents
HUMAN GENE THERAPY
2005; 16 (2): 271-277
Induction of peripheral inflammation increases the expression of the Nav1.7 sodium channel in sensory neurons, potentially increasing their excitability. Peripheral inflammation also produces hyperalgesia in humans and an increase in nociceptive responsiveness in animals. To test the relationship between these two phenomena we applied a recombinant herpes simplex-based vector to the hindpaw skin of mice, which encoded both green fluorescent protein (GFP) as well as an antisense sequence to the Nav1.7 gene. The hindpaw was subsequently injected with complete Freund's adjuvant to induce robust inflammation. Application of the vector, but not a control vector encoding only GFP, prevented an increase in Nav1.7 expression in GFP-positive neurons and prevented development of hyperalgesia in both C and Adelta thermonociceptive tests. These results provide clear evidence of the involvement of an increased expression of the Nav1.7 channel in nociceptive neurons in the development of inflammatory hyperalgesia.
View details for Web of Science ID 000227543900012
View details for PubMedID 15761266
- The projectile tooth of a fish-hunting cone snail: Conus catus injects venom into fish prey using a high-speed ballistic mechanism BIOLOGICAL BULLETIN 2004; 207 (2): 77-79
A gastropod toxin selectively slows early transitions in the Shaker K channel's activation pathway
JOURNAL OF GENERAL PHYSIOLOGY
2004; 123 (6): 685-696
A toxin from a marine gastropod's defensive mucus, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT), was found to inhibit voltage-gated potassium channels by a novel mechanism. Voltage-clamp experiments with Shaker K channels reveal that externally applied BrMT slows channel opening but not closing. BrMT slows K channel activation in a graded fashion: channels activate progressively slower as the concentration of BrMT is increased. Analysis of single-channel activity indicates that once a channel opens, the unitary conductance and bursting behavior are essentially normal in BrMT. Paralleling its effects against channel opening, BrMT greatly slows the kinetics of ON, but not OFF, gating currents. BrMT was found to slow early activation transitions but not the final opening transition of the Shaker ILT mutant, and can be used to pharmacologically distinguish early from late gating steps. This novel toxin thus inhibits activation of Shaker K channels by specifically slowing early movement of their voltage sensors, thereby hindering channel opening. A model of BrMT action is developed that suggests BrMT rapidly binds to and stabilizes resting channel conformations.
View details for DOI 10.1085/jgp.200409047
View details for Web of Science ID 000221988000006
View details for PubMedID 15148327
Selective open-channel block of Shaker (Kv1) potassium channels by S-nitrosodithiothreitol (SNDTT)
JOURNAL OF GENERAL PHYSIOLOGY
2001; 118 (1): 113-133
Large quaternary ammonium (QA) ions block voltage-gated K(+) (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH(2)CH(OH)CH(OH)CH(2)SNO) produces qualitatively similar "open-channel block" in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na(+) or Ca(2)+ channels. Inactivation-removed ShakerB (ShBDelta) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose-response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K(d2) = 0.14 mM) is lower than that of the first molecule (K(d1) = 0.67 mM), indicating cooperativity. The half-blocking concentration (K(1/2)) is approximately 0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.
View details for Web of Science ID 000169782800010
View details for PubMedID 11429448
Role of prey-capture experience in the development of the escape response in the squid Loligo opalescens: A physiological correlate in an identified neuron
JOURNAL OF EXPERIMENTAL BIOLOGY
2000; 203 (3): 559-565
Although extensively used for biophysical studies, the squid giant axon system remains largely unexplored in regard to in vivo function and modulation in any biologically relevant context. Here we show that successful establishment of the recruitment pattern for the giant axon in the escape response elicited by a brief electrical stimulus depends on prey-capture experience early in life. Juvenile squid fed only slow-moving, easy-to-capture prey items (Artemia salina) develop deficits in coordinating activity in the giant axon system with that of a parallel set of non-giant motor axons during escape responses. These deficits are absent in cohorts fed fast-moving, challenging prey items (copepods). These results suggest that the acquisition of inhibitory control over the giant axon system is experience-dependent and that both prey-capture and escape behavior depend on this control.
View details for Web of Science ID 000085498100013
View details for PubMedID 10637184
A family of delayed rectifier Kv1 cDNAs showing cell type-specific expression in the squid stellate ganglion giant fiber lobe complex
JOURNAL OF NEUROSCIENCE
1997; 17 (13): 5070-5079
Squid giant axons are formed by giant fiber lobe (GFL) neurons of the stellate ganglion (SG). Other large motoneurons in the SG form a parallel system. A small family of cDNAs (SqKv1A-D) encoding Kv1 alpha-subunits was identified in a squid (Loligo opalescens) SG/GFL library. Members have distinct 5' untranslated regions (UTRs) and initial coding regions, but beyond a certain point (nucleotide 34 of SqKv1A) only nine differences exist. 3' UTRs are identical. Predicted alpha-subunits are nearly identical, and only the N termini differ significantly, primarily in length. RNase protection assays that use RNA isolated from specific SG regions show that SqKv1A mRNA is expressed prominently in the GFL but not in the SG proper. SqKv1B yields the opposite pattern. SqKv1D also is expressed only in the SG. SqKv1C expression was not detectable. In situ hybridizations confirm these results and reveal that SqKv1B mRNA is abundant in many large neurons of the SG, whereas SqKv1D expression is limited to small isolated clusters of neurons. SqKv1A and B are thus the predominant Kv1 mRNAs in the SG/GFL complex. Activation properties of SqKv1A and B channels expressed in oocytes are very similar to one another and compare favorably with properties of native delayed rectifier channels in GFL neurons and large SG neurons. The Kv1 complement in these squid neurons thus seems to be relatively simple. Several differences exist between cloned and native channels, however, and may reflect differences in the cellular environments of oocytes and neurons.
View details for Web of Science ID A1997XE95200016
View details for PubMedID 9185544
Fast and slow activation kinetics of voltage-gated sodium channels in molluscan neurons
JOURNAL OF NEUROPHYSIOLOGY
1997; 77 (5): 2373-2384
Whole cell patch-clamp recordings of Na current (I(Na)) were made under identical experimental conditions from isolated neurons from cephalopod (Loligo, Octopus) and gastropod (Aplysia, Pleurobranchaea, Doriopsilla) species to compare properties of activation gating. Voltage dependence of peak Na conductance (gNa) is very similar in all cases, but activation kinetics in the gastropod neurons studied are markedly slower. Kinetic differences are very pronounced only over the voltage range spanned by the gNa-voltage relation. At positive and negative extremes of voltage, activation and deactivation kinetics of I(Na) are practically indistinguishable in all species studied. Voltage-dependent rate constants underlying activation of the slow type of Na channel found in gastropods thus appear to be much more voltage dependent than are the equivalent rates in the universally fast type of channel that predominates in cephalopods. Voltage dependence of inactivation kinetics shows a similar pattern and is representative of activation kinetics for the two types of Na channels. Neurons with fast Na channels can thus make much more rapid adjustments in the number of open Na channels at physiologically relevant voltages than would be possible with only slow Na channels. This capability appears to be an adaptation that is highly evolved in cephalopods, which are well known for their high-speed swimming behaviors. Similarities in slow and fast Na channel subtypes in molluscan and mammalian neurons are discussed.
View details for Web of Science ID A1997WZ56300011
View details for PubMedID 9163364
Fast inactivation of delayed rectifier K conductance in squid giant axon and its cell bodies
JOURNAL OF GENERAL PHYSIOLOGY
1997; 109 (4): 435-448
Inactivation of delayed rectifier K conductance (gk) was studied in squid giant axons and in the somata of giant fiber lobe (GFL) neurons. Axon measurements were made with an axial wire voltage clamp by pulsing to VK (approximately -10 mV in 50-70 mM external K) for a variable time and then assaying available gK with a strong, brief test pulse. GFL cells were studied with whole-cell patch clamp using the same prepulse procedure as well as with long depolarizations. Under our experimental conditions (12-18 degrees C, 4 mM internal MgATP) a large fraction of gK inactivates within 250 ms at -10 mV in both cell bodies and axons, although inactivation tends to be more complete in cell bodies. Inactivation in both preparations shows two kinetic components. The faster component is more temperature-sensitive and becomes very prominent above 12 degrees C. Contribution of the fast component to inactivation shows a similar voltage dependence to that of gK, suggesting a strong coupling of this inactivation path to the open state. Omission of internal MgATP or application of internal protease reduces the amount of fast inactivation. High external K decreases the amount of rapidly inactivating IK but does not greatly alter inactivation kinetics. Neither external nor internal tetraethylammonium has a marked effect on inactivation kinetics. Squid delayed rectifier K channels in GFL cell bodies and giant axons thus share complex fast inactivation properties that do not closely resemble those associated with either C-type or N-type inactivation of cloned Kvl channels studied in heterologous expression systems.
View details for Web of Science ID A1997WT51900004
View details for PubMedID 9101403
All-or-none contraction and sodium channels in a subset of circular muscle fibers of squid mantle
1996; 191 (3): 337-340
Motor function in squid (Loligo) mantle reflects the highly coordinated activity of two motor pathways associated with giant and non-giant motor axons that respectively produce all-or-none and graded contractions in mantle muscle. Whereas both types of axons innervate circular mantle muscle fibers, precise nerve-muscle relationships remain unclear. Are squid like most invertebrates, in which single muscle fibers receive dual innervation from giant and non-giant motor axons, or is squid mantle configured more like vertebrates, in which parallel motor axon systems innervate distinct fast and slow muscle fibers? In this report, we describe giant and nongiant motor pathways that appear to control different pools of circular muscle fibers in squid. A subset of circular muscle fibers possesses large Na currents, and these fibers are proposed to employ Na-dependent action potentials to produce fast, all-or-none muscle twitches associated with giant axon stimulation.
View details for Web of Science ID A1996WA83400001
View details for PubMedID 8976593
Molecular identification of SqKv1A - A candidate for the delayed rectifier K channel in squid giant axon
JOURNAL OF GENERAL PHYSIOLOGY
1996; 108 (3): 207-219
We have cloned the cDNA for a squid Kvl potassium channel (SqKv1A). SqKv1A mRNA is selectively expressed in giant fiber lobe (GFL) neurons, the somata of the giant axons. Western blots detect two forms of SqKv1A in both GFL neuron and giant axon samples. Functional properties of SqKv1A currents expressed in Xenopus oocytes are very similar to macroscopic currents in GFL neurons and giant axons. Macroscopic K currents in GFL neuron cell bodies, giant axons, and in Xenopus oocytes expressing SqKv1A, activate rapidly and inactivate incompletely over a time course of several hundred ms. Oocytes injected with SqKv1A cRNA express channels of two conductance classes, estimated to be 13 and 20 pS in an internal solution containing 470 mM K. SqKv1A is thus a good candidate for the "20 pS" K channel that accounts for the majority of rapidly activating K conductance in both GFL neuron cell bodies and the giant axon.
View details for Web of Science ID A1996VF64200008
View details for PubMedID 8882864
AMINO-ACID-SEQUENCE OF A PUTATIVE SODIUM-CHANNEL EXPRESSED IN THE GIANT-AXON OF THE SQUID LOLIGO-OPALESCENS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1993; 90 (21): 10026-10030
A full-length cDNA encoding a putative Na+ channel (GFLN1) has been cloned from a library prepared from the stellate ganglion of Loligo opalescens. The cDNA encodes a predicted protein of 1784 amino acids. Regions of the GFLN1 protein with defined functional importance (membrane span S4, the SS1 and SS2 segments, and interdomain III-IV) are highly conserved among all vertebrate Na+ channel alpha-subunit structures. Northern blot hybridization and RNase protection assays verify that mRNA corresponding to GFLN1 is expressed in neurons of the giant fiber lobe that form the giant axon. We propose that GFLN1 encodes the Na+ channel that has been extensively studied in the squid axon.
View details for Web of Science ID A1993MF29600059
View details for PubMedID 8234251
ELECTRICAL RESPONSES TO CHEMICAL-STIMULATION OF SQUID OLFACTORY RECEPTOR-CELLS
JOURNAL OF EXPERIMENTAL BIOLOGY
1992; 162: 231-249
View details for Web of Science ID A1992HD81100014
CONTROL OF THE SPATIAL-DISTRIBUTION OF SODIUM-CHANNELS IN GIANT FIBER LOBE NEURONS OF THE SQUID
1990; 5 (5): 663-674
Na+ channels are present at high density in squid giant axon but are absent from its somata in the giant fiber lobe (GFL) of the stellate ganglion. GFL cells dispersed in vitro maintain growing axons and develop a Na+ channel distribution similar to that in vivo. Tunicamycin, a glycosylation inhibitor, selectively disrupts the spatially appropriate, high level expression of Na+ channels in axonal membrane but has no effect on expression in cell bodies, which show low level, inappropriate expression in vitro. This effect does not appear to involve alteration in Na+ channel turnover or axon viability. K+ channel distribution is unaffected. Thus, glycosylation appears to be involved in controlling Na+ channel localization in squid neurons.
View details for Web of Science ID A1990EJ86800010
View details for PubMedID 2171590
JET-PROPELLED ESCAPE IN THE SQUID LOLIGO-OPALESCENS - CONCERTED CONTROL BY GIANT AND NON-GIANT MOTOR AXON PATHWAYS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1990; 87 (8): 2911-2915
Recordings of stellar nerve activity were made during escape responses in living squid. Short-latency activation of the giant axons is triggered by light-flash stimulation that elicits a stereotyped startle-escape response and powerful jet. Many other types of stimuli produce a highly variable, delayed-escape response with strong jetting primarily controlled by a small axon motor pathway. In such cases, activation of the giant axons is not necessary for a vigorous escape jet. When they are utilized, the giant axons are not activated until well after the non-giant system initiates the escape response, and excitation is critically timed to boost the rise in intramantle pressure. Squid thus show at least two escape modes in which the giant axons can contribute in different ways to the control of a highly flexible behavior.
View details for Web of Science ID A1990CZ29900010
View details for PubMedID 2326255