Forward shift of feeding buzz components of dolphins and belugas during associative learning reveals a likely connection to reward expectation, pleasure and brain dopamine activation.

For many years, we heard sounds associated with reward from dolphins and belugas. We named these pulsed sounds victory squeals (VS), as they remind us of a child's squeal of delight. Here we put these sounds in context with natural and learned behavior. Like bats, echolocating cetaceans produce feeding buzzes as they approach and catch prey. Unlike bats, cetaceans continue their feeding buzzes after prey capture and the after portion is what we call the VS. Prior to training (or conditioning), the VS comes after the fish reward; with repeated trials it moves to before the reward. During training, we use a whistle or other sound to signal a correct response by the animal. This sound signal, named a secondary reinforcer (SR), leads to the primary reinforcer, fish. Trainers usually name their whistle or other SR a bridge, as it bridges the time gap between the correct response and reward delivery. During learning, the SR becomes associated with reward and the VS comes after the SR rather than after the fish. By following the SR, the VS confirms that the animal expects a reward. Results of early brain stimulation work suggest to us that SR stimulates brain dopamine release, which leads to the VS. Although there are no direct studies of dopamine release in cetaceans, we found that the timing of our VS is consistent with a response after dopamine release. We compared trained vocal responses to auditory stimuli with VS responses to SR sounds. Auditory stimuli that did not signal reward resulted in faster responses by a mean of 151 ms for dolphins and 250 ms for belugas. In laboratory animals, there is a 100 to 200 ms delay for dopamine release. VS delay in our animals is similar and consistent with vocalization after dopamine release. Our novel observation suggests that the dopamine reward system is active in cetacean brains.

An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites.

Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids. Limited digestion and gamma irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA. No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids. Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.

The in vivo conformation of the plastid DNA of Toxoplasma gondii: implications for replication.

The Phylum Apicomplexa comprises thousands of obligate intracellular parasites, some of which cause serious disease in man and other animals. Though not photosynthetic, some of them, including the malaria parasites (Plasmodium spp.) and the causative organism of Toxoplasmosis, Toxoplasma gondii, possess a remnant plastid partially determined by a highly derived residual genome encoded in 35 kb DNA. The genetic maps of the plastid genomes of these two organisms are extremely similar in nucleotide sequence, gene function and gene order. However, a study using pulsed field gel electrophoresis and electron microscopy has shown that in contrast to the malarial version, only a minority of the plastid DNA of Toxoplasma occurs as circular 35 kb molecules. The majority consists of a precise oligomeric series of linear tandem arrays of the genome, each oligomer terminating at the same site in the genetic map, i.e. in the centre of a large inverted repeat (IR) which encodes duplicated tRNA and rRNA genes. This overall topology strongly suggests that replication occurs by a rolling circle mechanism initiating at the centre of the IR, which is also the site at which the linear tails of the rolling circles are processed to yield the oligomers. A model is proposed which accounts for the quantitative structure of the molecular population. It is relevant that a somewhat similar structure has been reported for at least three land plant chloroplast genomes.

Complete gene map of the plastid-like DNA of the malaria parasite Plasmodium falciparum.

Malaria parasites, and other parasitic protists of the Phylum Apicomplexa, carry a plastid-like genome with greatly reduced sequence complexity. This 35 kb DNA circle resembles the plastid DNA of non-photosynthetic plants, encoding almost exclusively components involved in gene expression. The complete gene map described here includes genes for duplicated large and small subunit rRNAs, 25 species of tRNA, three subunits of a eubacterial RNA polymerase, 17 ribosomal proteins, and a translation elongation factor. In addition, it codes for an unusual member of the Clp family of chaperones, as well as an open reading frame of unknown function found in red algal plastids. Transcription is polycistronic. This plastid-like DNA molecule is conserved in several genera of apicomplexans and is conjectured to have been acquired by an early progenitor of the Phylum by secondary endosymbiosis. The function of the organelle (plastid) carrying this DNA remains obscure, but appears to be specified by genes transferred to the nucleus.

Phylogenetic analysis of the rpoB gene from the plastid-like DNA of Plasmodium falciparum.

Malaria and other Apicomplexan parasites harbour two extrachromosomal DNAs. One is mitochondrial and the other is a 35-kb circle with some plastid-like features but whose provenance and function is unknown. In addition to genes for rRNAs, tRNAs and ribosomal proteins, the 35-kb circular DNA of Plasmodium falciparum carries an rpoBC operon which encodes subunits of a eubacteria-like RNA polymerase. The phylogenetic analysis of the complete rpoB sequence presented here supports our inference that the 35-kb circle is the remnant of a plastid genome.

Technetium-99 in generator systems.

Technetium-99m solutions always contain Tc-99. The amount varies considerably, depending on production methods and conditions, generator performance, and the time between production or separation and use. There is increasing evidence that labeling and imaging with some radiopharmaceutical kits is adversely affected when the Tc-99 exceeds certain amounts. The sensitivity of particular kits to Tc-99 depends on the ligand, the amount of usable Sn2+, and the ratio of Tc to ligand. Although Tc-99 formed during production is removed in the final steps of generator manufacture, some may appear in early extractions in the solvent extraction process. If elution or extraction efficiencies are high, any Tc-99 is reduced within about two elutions to an insignificant level that is maintained in subsequent elutions. If efficiencies are below about 40%, however, the ratio of Tc-99 to Tc-99m increases with each elution.

A case series of patients with lamotrigine toxicity at one center from 2003 to 2012.

INTRODUCTION: Lamotrigine is a phenyltriazine compound that inhibits voltage-gated sodium channels, decreasing release of glutamate and aspartate, and inhibits serotonin, norepinephrine and dopamine reuptake. Reports of toxicity in the literature are limited to case reports and primarily involve coingestants. This case series is intended to report the clinical manifestations of lamotrigine toxicity. METHODS: This retrospective case series from 2003 to 2012 studies the effects of lamotrigine toxicity when not confounded by coingestants. Admission records at an inpatient toxicology center were reviewed for lamotrigine-only exposure based on history with supporting laboratory data when available. After identification, these charts were reviewed again to characterize vital signs, neurological examination findings, specific laboratory and electrocardiography parameters, and complications. RESULTS: Fifty-seven patients were identified with possible lamotrigine toxicity. Nine patients, including three toddlers, had lamotrigine-only ingestions. Three of these patients had seizures, four were hypertensive, five were tachycardic, and four experienced tachypnea. Mental status was altered in all nine (depressed (n = 4), agitated (n = 5) or both (n = 3)). Five patients were hyperreflexic and experienced intermittent myoclonus, and two had inducible clonus. On electrocardiogram, two patients experienced QRS prolongation (114-116 ms), and four had QTc prolongation (463-586 ms). No patient had life-threatening symptoms or signs. Serum levels of lamotrigine were available in seven patients, and averaged 35.4 mg/L (17-90 mg/L). The therapeutic range for sLTG is 3-14 mg/L. CONCLUSIONS: Lamotrigine toxicity manifested with minor-moderate neurologic and/or electrocardiographic effects. Toxicity reflects the known pharmacologic actions of lamotrigine: serotonin, norepinephrine and dopamine reuptake inhibition, and sodium channel blockade.

Reversible mechanical actuation of elastomeric nanopores.

Mechanical resizing of individual nanopores in a thermoplastic polyurethane elastomer has been characterized. Specimen nanopores were conical, with smaller hole dimensions of the order of tens to hundreds of nanometres. Electrophoretic current measurements show that the estimated nanopore radius can be reversibly actuated over an order of magnitude by stretching and relaxing the elastomer. Within a working range of stretching, current is proportional to specimen extension to the power of a constant, n, which ranges from 0.9 to 2.3 for different specimens. The data indicate that scaling of the effective pore radius is super-affine. At strains below the working range, the pore size is relatively unresponsive to stretching. Macroscopic elastomer extension has been related to local radial strain (50-250 µm from the pore) using optical microscopy. Scanning electron microscopy and atomic force microscopy have been used to observe membrane surface features.

Receiving beam patterns and directivity indices of the Atlantic bottlenose dolphin Tursiops truncatus.

The receiving beam patterns of an Atlantic bottlenose dolphin was measured in both the vertical and horizontal planes for frequencies of 30, 60, and 120 kHz. Measurements in the vertical plane were performed by training the dolphin to rotate on its side and station on a vertically oriented biteplate device. A signal source was positioned 3.5 m directly in front of the animal on an arc while the position of a broadband noise source was varied in azimuth along the arc, with the animal stationed at the origin of the arc. Using a go/no-go response procedure, the masked threshold of the dolphin was determined by varying the intensity of the noise source via a tracking method of stimulus presentation, for different azimuth's of the noise source. Measurements in the horizontal plane were obtained with the dolphin stationing on a horizontal biteplate. Two masking noise sources, projecting equal levels of uncorrelated noise, were located at +/- 20 degrees on either side of the arc midpoint. The levels of the noise source were held constant and the masked thresholds determined by varying the level of the signal source while positioned at different azimuths along the arc. Results indicated that maximum sensitivity in the vertical plane (the major axis of the beam) occurred between 5 degrees and 10 degrees above the midline of the animal's mouth and that the beam patterns were not symmetrical about the major axis. The sensitivity dropped off more quickly with increasing angle above the head than below. The beam patterns in the horizontal plane were directed forward and were fairly symmetrical about the midline of the animal's body.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of complex echoes in noise by an echolocating dolphin.

Dolphins echolocate with short broadband acoustic signals that have good time resolution properties. Received echoes are often complex, with many resolvable highlights or components caused by reflection of the incident signal from external and internal boundaries of a target and from different propagational modes within a target. A series of experiments was performed to investigate how dolphins perceive complex echoes. Echoes were produced by a microprocessor-controlled electronic target simulator that captured each emitted click and retransmitted the signal back to the animal after an appropriate time delay. The use of this "phantom" target allowed for precise control of the number of highlights, the time separation between highlights, and the relative amplitudes of highlights in the simulated echoes. An echolocating dolphin was trained to perform a target detection task in the presence of masking noise using these phantom echoes. The properties of simulated echoes were systematically varied, and corresponding shifts in the dolphin's detection threshold were observed, allowing for inferences of how the dolphin perceived echoes. The dolphin performed like an energy detector with an integration time of approximately 264 microseconds.

Echolocation transmitting beam of the Atlantic bottlenose dolphin.

The transmitting beam patterns of echolocation signals emitted by an Atlantic bottlenose dolphin Tursiops truncatus were measured in the vertical and horizontal planes with an array of seven hydrophones. Particular emphasis was placed on accurately verifying the animal's position on a bite-plate/tail-rest stationing device using underwater video monitoring equipment. The major axis of the vertical beam was directed at an angle of 5 degrees above the plane defined by the animal's lips. This angle was 15 degrees lower than previously measured. The vertical beam measurements indicate that the major axis of the transmitting beam is aligned with the major axis of the receiving beam. The horizontal beam was directed forward. The directivity index of 26.5 dB calculated from the beam pattern measured in both planes agreed well with previous calculation of 25.4 dB.

Assessment of dolphin (Tursiops truncatus) auditory sensitivity and hearing loss using jawphones.

Devices known as jawphones have previously been used to measure interaural time and intensity discrimination in dolphins. This study introduces their use for measuring hearing sensitivity in dolphins. Auditory thresholds were measured behaviorally against natural background noise for two bottlenose dolphins (Tursiops truncatus); a 14-year-old female and a 33-year-old male. Stimuli were delivered to each ear independently by placing jawphones directly over the pan bone of the dolphin's lower jaw, the assumed site of best reception. The shape of the female dolphin's auditory functions, including comparison measurements made in the free field, favorably matches that of the accepted standard audiogram for the species. Thresholds previously measured for the male dolphin at 26 years of age indicated a sensitivity difference between the ears of 2-3 dB between 4-10 kHz, which was considered unremarkable at the time. Thresholds for the male dolphin reported in this study suggest a high-frequency loss compared to the standard audiogram. Both of the male's ears have lost sensitivity to frequencies above 55 kHz and the right ear is 16-33 dB less sensitive than the left ear over the 10-40 kHz range, suggesting that males of the species may lose sensitivity as a function of age. The results of this study support the use of jawphones for the measurement of dolphin auditory sensitivity.

Classification of dolphin echolocation clicks by energy and frequency distributions.

Dolphins demonstrate an adaptive control over echolocation click production, but little is known of the manner or degree with which control is exercised. Echolocation clicks (N approximately 30,000) were collected from an Atlantic bottlenose dolphin (Tursiops truncatus) performing object discrimination tasks in order to investigate differential click production. Seven categories of clicks were identified using the spectral conformation and relative position of -3 and -10 dB peaks. A counterpropagation network utilizing 16 inputs, 50 hidden units, and 8 output units was trained to classify clicks using the same spectral variables. The network classified novel clicks with 92% success. Additional echolocation clicks (N > 24,000) from two other dolphins were submitted to the network for classification. Classified echolocation clicks were analyzed for animal specific differences, changes in predominant click type within click trains, and task-related specificity. Differences in animal and task performance may influence click type and click train length.

The critical interval in dolphin echolocation: what is it?

A backward masking function relating target detection to masker delay was generated for a bottlenosed dolphin in an active echolocation target detection task. The masker was triggered by each outgoing echolocation click and could be temporally adjusted from coincidence with the target echo to delays of 700 microseconds. The animal reported target condition using a go/no-go response procedure. A modified method of constants was used to present the four masking delay intervals. Results indicated that 700- and 500-microseconds delays had little effect on target detection. However, as the delay was reduced to 100 microseconds, detection dropped to chance performance. The calculated 70% detection threshold corresponded to a delay of 265 microseconds. The results are discussed in support of the view that time separation pitch (TSP) may be an analytic mechanism used by the dolphin to discern various within-echo target attributes rather than an analytic mechanism for determining target range.

Zirconium molybdate gel as a generator for technetium-99m--I. The concept and its evaluation.

A new 99mTc generator for the preparation of radiopharmaceuticals has been developed. Its elution characteristics and chemical stability merit its consideration as a replacement for present generators. The 99Mo is present as a zirconium molybdate gel, the high molybdate content of which allows the use of (n, gamma) 99Mo. The resulting generator is as effective and as convenient to use as chromatographic generators that are based on fission product 99Mo. Laboratory evaluations of the concept at low levels of radioactivity described in this paper show that the gel can be prepared in a form which is stable when used in a generator and from which TcO4- can be eluted in yields of 80-85%. The influence of formulation variables on gel properties was established. The performance of this generator using 99Mo with specific activities between 4 and 13 GBq g-1 is described in a further paper.

Signal processing of the echo signatures returned by submerged shells insonified by dolphin "clicks:" active classification.

A large set of dolphin-emitted acoustic pulses ("echolocation clicks") have been examined, which were reflected from various elastic shells that were suspended, underwater, 4.5 m in front of the animal in a large test site in Kaneohe Bay, Hawaii. A carefully instrumented analog-to-digital system continuously captured the emitted clicks and also the returned, backscattered echoes (A/D conversion at 500 kHz). Using standard conditioning techniques and food reinforces, the dolphin is taught to push an underwater paddle when the "correct" target-the one he has been trained to identify-is presented to him. He communicates his consistently correct identifying choices in this manner. Many echoes returned by three types of cylindrical shells in both the time and frequency domains as well as in the joint time-frequency (t-f) domain, by means of Wigner-type distributions have been examined. It will be shown exactly how specific features observable in these displays are directly related to the physical characteristics of the shells. This processing takes advantage of certain fundamental resonance principles to show which echo features contain information about the size, shape, wall thickness, and material composition of both the shell and its filler substance. In the same fashion that these resonance features give the identifying characteristics of each shell, it is believed they may also give them to the dolphin. These echo features may allow him to extract the target properties by inspection without any need for computations. It is claimed that this may be the fundamental physical explanation of the dolphin's amazing target ID feats, upon which they base their recognition choices. This claim may be substantiated by the detailed analysis of many typical echoes returned by various shells, when they are interrogated by several dolphins. Thus far, this analysis of many echoes from many shells has only been carried out for a single dolphin.

Zirconium molybdate gel as a generator for technetium-99m--II. High activity generators.

The characteristics of zirconium molybdate gels were described in Part I of this paper. Here it is shown that zirconium molybdate gel, prepared from neutron irradiated molybdenum oxide with 99Mo specific activities between 4.3 and 13.5 GBq g-1 is sufficiently stable for use as a matrix in preparation of high activity 99mTc generators. Elution efficiencies of generators containing from 2 to 6 g of gel ranged from 83 to 50% for 10 mL elutions. Generators containing 150 g of gel were eluted with efficiencies of 80-98%, with the activity peak in the first 50 mL. Radionuclidic impurities were considerably below the limits set by the British Pharmacopoeia. Although the pH of eluates was less than 4, subsequent treatment with a small zirconium oxide bed provided adjustment to within BP limits, and reduced 99Mo impurity levels tenfold to values near 10(-4)%. No significant differences were found between the biodistributions in rats of radiopharmaceuticals prepared with pertechnetate from gel generators and those from other generators.