State-changes in the swimmeret system: a neural circuit that drives locomotion.

The crayfish swimmeret system undergoes transitions between a silent state and an active state. In the silent state, no patterned firing occurs in swimmeret motor neurons. In the active state, bursts of spikes in power stroke motor neurons alternate periodically with bursts of spikes in return stroke motor neurons. In preparations of the isolated crayfish central nervous system (CNS), the temporal structures of motor patterns expressed in the active state are similar to those expressed by the intact animal. These transitions can occur spontaneously, in response to stimulation of command neurons, or in response to application of neuromodulators and transmitter analogues. We used single-electrode voltage clamp of power-stroke exciter and return-stroke exciter motor neurons to study changes in membrane currents during spontaneous transitions and during transitions caused by bath-application of carbachol or octopamine (OA). Spontaneous transitions from silence to activity were marked by the appearance of a standing inward current and periodic outward currents in both types of motor neurons. Bath-application of carbachol also led to the development of these currents and activation of the system. Using low Ca(2+)-high Mg(2+) saline to block synaptic transmission, we found that the carbachol-induced inward current included a direct response by the motor neuron and an indirect component. Spontaneous transitions from activity to silence were marked by disappearance of the standing inward current and the periodic outward currents. Bath-application of OA led promptly to the disappearance of both currents, and silenced the system. OA also acted directly on both types of motor neurons to cause a hyperpolarizing outward current that would contribute to silencing the system.

Limb movements during locomotion: Tests of a model of an intersegmental coordinating circuit.

During normal forward swimming, the swimmerets on neighboring segments of the crayfish abdomen make periodic power-stroke movements that have a characteristic intersegmental difference in phase. Three types of intersegmental interneurons that originate in each abdominal ganglion are necessary and sufficient to maintain this phase relationship. A cellular model of the intersegmental coordinating circuit that also produces the same intersegmental phase has been proposed. In this model, coordinating axons synapse with local interneurons in their target ganglion and form a concatenated circuit that links neighboring segmental ganglia. This model assumed that coordinating axons projected to their nearest-neighboring ganglion but not farther. We tested this assumption in two sets of experiments. If the assumption is correct, then blocking synaptic transmission in an intermediate ganglion should uncouple swimmeret activity on opposite sides of the block. We bathed individual ganglia in a low Ca(2+)-high Mg(2+) saline that effectively silenced both motor output from the ganglion and the coordinating interneurons that originated in it. With this block in place, other ganglia on opposite sides of the block could nonetheless maintain their normal phase difference. Simultaneous recordings of spikes in coordinating axons on opposite sides of the blocked ganglion showed that these axons projected beyond the neighboring ganglion. Selective bilateral ablation of the tracts in which these axons ran showed that they were necessary and usually sufficient to maintain coordination across a blocked ganglion. We discuss revisions of the cellular model of the coordinating circuit that would incorporate these new results.

Functional organization of crayfish abdominal ganglia. III. Swimmeret motor neurons.

Swimmerets are limbs on several segments of the crayfish abdomen that are used for forward swimming and other behaviors. We present evidence that the functional modules demonstrated previously in physiological experiments are reflected in the morphological disposition of swimmeret motor neurons. The single nerve that innervates each swimmeret divides into two branches that separately contain the axons of power-stroke and return-stroke motor neurons. We used Co(++) or biocytin to backfill the entire pool of neurons that innervated a swimmeret, or functional subsets whose axons occurred in particular branches. Each filled cell body extended a single neurite that projected first to the Lateral Neuropil (LN), and there branched to form dendritic structures and its axon. All the motor neurons that innervated one swimmeret had cell bodies located in the ganglion from which their axons emerged, and the cell bodies of all but two of these neurons were located ipsilateral to their swimmeret. Counts of cell bodies filled from selected peripheral branches revealed about 35 power-stroke motor neurons and 35 return-stroke motor neurons. The cell bodies of these two types were segregated into different clusters within the ganglion, but both types sent their neurites into the ipsilateral LN and had their principle branches in this neuropil. We saw no significant differences in the numbers or distributions of these motor neurons in ganglia A2 through A5. These anatomical features are consistent with the physiological evidence that each swimmeret is controlled by its own neural module, which drives the alternating bursts of impulses in power-stroke and return-stroke motor neurons. We propose that the LN is the site of the synaptic circuit that generates this pattern.

Intersegmental coordination of swimmeret movements: mathematical models and neural circuits.

Swimmerets move periodically through a cycle of power-strokes and return-strokes. Swimmerets on neighboring segments differ in phase by approximately 25%, and maintain this difference even when the period of the cycle changes from < 1 to > 4 Hz. We constructed a minimal cellular model of the segmental pattern-generating circuit which incorporated its essential components, and whose dynamics were like those of the local circuit. Three different intersegmental coordinating units were known to link neighboring ganglia, but their targets are unknown. We constructed different intersegmental circuits which these units might form between neighboring cellular models, and compared their dynamics with the real system. One intersegmental circuit could maintain an approximately 25% phase difference through a range of periods. In physiological experiments, we identified three types of intersegmental interneurons that originate in each ganglion and project to its neighbors. These neurons fire bursts at certain parts of the swimmeret cycle in their home ganglion. These three neurons are necessary and sufficient to maintain normal coordination between neighboring segments. Their properties conform to the predictions of the cellular model.

Modulation of force during locomotion: differential action of crustacean cardioactive peptide on power-stroke and return- stroke motor neurons.

Crustacean cardioactive peptide (CCAP) elicited expression of the motor pattern that drives coordinated swimmeret beating in crayfish and modulated this pattern in a dose-dependent manner. In each ganglion that innervates swimmerets, neurons with CCAP-like immunoreactivity sent processes to the lateral neuropils, which contain branches of swimmeret motor neurons and the local pattern-generating circuits. CCAP affected each of the four functional groups of motor neurons, power-stroke excitors (PSE), return-stroke excitors (RSE), power-stroke inhibitors (PSI), and return-stroke inhibitors (RSI), that innervate each swimmeret. When CCAP was superfused, the membrane potentials of these neurons began to oscillate periodically about their mean potentials. The mean potentials of PSE and RSI neurons depolarized, and some of these neurons began to fire during each depolarization. Both intensity and durations of PSE bursts increased significantly. The mean potentials of RSE and PSI neurons hyperpolarized, and these neurons were less likely to fire during each depolarization. When CCAP was superfused in a low Ca2+ saline that blocked chemical transmission, these changes in mean potential persisted, but the periodic oscillations disappeared. These results are evidence that CCAP acts at two levels: activation of local premotor circuits and direct modulation of swimmeret motor neurons. The action on motor neurons is differential; PSEs and RSIs are excited, but RSEs and PSIs are inhibited. The consequences of this selectivity are to increase intensity of bursts of impulses that excite power-stroke muscles.

Identification and characterization of a new and distinct molecular subtype of human T-cell lymphotropic virus type 2.

Molecular studies have demonstrated the existence of at least two major subtypes of human T-cell lymphotropic virus type 2 (HTLV-2), designated HTLV-2a and HTLV-2b. To further investigate the heterogeneity of this family of viruses, we have characterized the HTLV-2 subtypes present in several urban areas in Brazil. DNAs from peripheral blood mononuclear cells of a large number of infected individuals, the majority of whom were intravenous drug abusers, were analyzed by using PCR with restriction fragment length polymorphism and nucleotide sequencing analysis. Restriction fragment length polymorphism analysis of the env region suggested that all individuals were infected with the HTLV-2a subtype, and this was confirmed by nucleotide sequence analysis. In contrast, nucleotide sequence analysis of the long terminal repeat demonstrated that although the viruses were more related to the HTLV-2a than to the HTLV-2b subtype, they clustered in a distinct phylogenetic group, suggesting that they may represent a new and distinct molecular subtype of HTLV-2. This conclusion was supported by nucleotide sequence analysis of the pX region, which demonstrated that the Tax proteins of the Brazilian viruses differed from that of prototype HTLV-2a isolates but were more similar to that of HTLV-2b in that they would be expected to have an additional 25 amino acids at the carboxy terminus. In transient expression assays, the extended Tax protein of the prototype HTLV-2a subtype. The studies suggest that the Brazilian viruses analyzed in this study, while being phylogenetically related to the prototypic HTLV-2a seen in North America, are phenotypically more related to HTLV-2b and can be justifiably classified as a new molecular subtype, which has been tentatively designated HTLV-2c.

Proctolin and excitation of the crayfish swimmeret system.

The ventral nerve cord of crayfish contains axons of five pairs of excitatory interneurons, each of which can activate the swimmeret system. Perfusion of the ventral nerve cord with the neuropeptide proctolin also activates the swimmeret system. The experiments reported here were conducted to test the hypothesis that one or more of these excitatory interneurons uses proctolin as a transmitter. Each of the five excitatory axons was located and stimulated separately in an individual crayfish, and similar motor activity was elicited by stimulating each of them. Quantitative comparison of spontaneous swimmeret motor patterns with activity caused by stimulating one of these excitatory axons, EC, or by perfusing with proctolin solutions showed that the motor patterns produced under these three conditions were not significantly different (P > 0.05). By using a new, affinity-purified proctolin antiserum, we labeled axons in the connective tissue between the last thoracic and first abdominal ganglion and compared the positions of labeled axons with the previously described positions of the excitatory axons. About 0.3% of the axons in these connective tissues showed proctolin-like immunoreactivity, but heavily labeled pairs of axons did occur bilaterally in the regions of excitatory swimmeret axons. The projections of these labeled axons into the abdominal ganglia were traced in serial plastic sections. Labeled processes were abundant in the lateral neuropils, the loci of the swimmeret pattern-generating circuitry. From this evidence, we propose that three of these excitatory swimmeret interneurons use proctolin as a transmitter, but that a fourth does not. The evidence for the fifth axon is ambiguous.

Neurons with histaminelike immunoreactivity in the segmental and stomatogastric nervous systems of the crayfish Pacifastacus leniusculus and the lobster Homarus americanus.

We used a polyclonal antiserum against histamine to map histaminelike immunoreactivity (HLI) in whole mounts of the segmental ganglia and stomatogastric ganglion of crayfish and lobster. Carbodiimide fixation permitted both HRP-conjugated and FITC-conjugated secondary antibodies to be used effectively to visualize HLI in these whole mounts. Two interneurons that send axons through the inferior ventricular nerve (ivn) and the stomatogastric nerve to the stomatogastric ganglion had strong HLI, both in crayfish and in lobster. These ivn interneurons were known from other evidence to be histaminergic. The neuropil of the stomatogastric ganglion in both crayfish and lobster contained brightly labeled terminals of axons that entered the ganglion from the stomatogastric nerve. No neuronal cell bodies in this ganglion had HLI. Each segmental ganglion contained at least one pair of neurons with HLI. Some neurons in the subesophageal ganglion and in each thoracic ganglion labeled very brightly. Axons of projection interneurons with strong HLI occurred in the dorsal lateral tracts of each segmental ganglion, and sent branches to the lateral neurophils and tract neurophils of each ganglion. All the labeled neurons were interneurons; no HLI was observed in peripheral nerves.

Intensive care experience with intravenous cimetidine.

This study reports the results of a retrospective review of the case records of 28 seriously ill patients who received intravenous cimetidine (generally 300 mg q8h) for the treatment of gastric discomfort and/or hemorrhage or for prophylaxis against stress-induced ulcers. Most of these patients presented with complex symptoms arising from a variety of pathological conditions including ischemic heart disease, myocardial infarction, cerebrovascular accident, pneumonia, and trauma. A number of patients also had acute gastrointestinal hemorrhage. Over two-thirds of the patients treated with intravenous cimetidine demonstrated a reduction in gastrointestinal symptom severity, and a statistically significant reduction in the mean severity rating for all patients was observed. Adverse reactions reported during cimetidine therapy were generally mild to moderate in severity and required discontinuance of therapy in only one patient. The most common complaint was headache. Intravenous cimetidine administered q8h offers a safe and cost-effective approach to H2-receptor blockade and reduction of gastric acid secretion in patients who are temporarily unable to take oral medication.

GABA-ergic neurons in the crayfish nervous system: an immunocytochemical census of the segmental ganglia and stomatogastric system.

We used an antiserum directed against gamma-aminobutyric acid (GABA) fixed with glutaraldehyde (Hoskins et al., Cell Tissue Res. 244:243-252, '86) to label neurons with GABA-like immunoreactivity (GLI) in wholemounts of the stomatogastric ganglion and each segmental ganglion of crayfish, except the brain. Each abdominal ganglion had an average of 63 labeled neurons, or 10% of all their neurons. Each peripheral nerve of each abdominal ganglion except the last contained labeled axons. Within each segment, the first peripheral nerve, N1, had five axons; the second peripheral nerve, N2, had at most four; and the third peripheral nerve, N3, had two. In the last ganglion, N2 had one labeled axon, N3 had two and N6 had two; the other nerves contained no labeled axons. A tabulation of the identified inhibitory neurons in the abdominal ganglia revealed that 40% of these GABA-ergic neurons have been identified. The subesophageal ganglion had many labeled neurons in clusters that formed a repeating pattern; it also had labeled neurons near its dorsal midline. The thoracic ganglia contained more labeled neurons than did the abdominals, but their patterns of labeling were similar. The commissural ganglia contained three clusters of labeled neurons and sent labeled axons to the esophageal ganglion. The esophageal ganglion contained four labeled neurons and many labeled axons. The stomatogastric ganglion contained labeled axon terminals but not labeled neurons.

Functional organization of crayfish abdominal ganglia: II. Sensory afferents and extensor motor neurons.

Abdominal ganglia of crayfish contain identifiable neuropils, commissures, longitudinal tracts, and vertical tracts. To determine the functional significance of this ganglionic framework, we backfilled the following types of neurons with cobalt chloride: sensory hair afferents, slow and fast extensor motor neurons, the segmental stretch receptor neurons, and their inhibitory accessory cells. After the cobalt ions were precipitated and intensified, we studied the central projections of the filled neurons within the ganglionic structures. All of the axons of these neurons exit or enter each of the first five abdominal ganglia through the second pair of nerves. Our description of the central projections of the hair afferents is the first in the literature. These afferents innervate the large ventral horseshoe neuropil (HN) in the core of each ganglion. This neuropil is homologous to the insect ventral association centers, which also process sensory information. Furthermore, we discovered that some of the crayfish afferents innervate glomeruli within the HN. The slow and fast extensor motor neurons, the stretch receptor neurons, and the accessory cells branch mostly in the dorsal part of the ganglion. We reinterpret previous identifications of the extensor neurons that were based largely on soma position. Together with our previous descriptions of the flexor motor neurons, these results allow us to relate both rapid tail-flips and slower postural movements to the structure of the segmental ganglia.

The PD programs: a method for the quantitative description of motor patterns.

We describe a family of 6 computer programs that measure, analyse and create graphic displays of complex motor patterns. The programs create lists of times at which successive bursts of impulses in different nerves started and stopped, and use these lists to calculate the periods and durations of these bursts and to calculate their phases relative to some specified frame of reference. When calculating phases, the programs take into account missing bursts or extra bursts in each reference interval. Individual programs then calculate descriptive statistics for these parameters, select lists of paired data for plotting and regression analysis, and prepare files for graphical display of statistics as boxplots. A final-program plots these files on a digital plotter. These programs are available for non-commercial use.

Functional organization of crayfish abdominal ganglia: I. The flexor systems.

For insect ganglia, Altman (Advances in Physiological Science, Vol. 23. Neurobiology of Invertebrates. New York: Pergamon Press, pp. 537-555, '81) proposed that individual neuropils control different motor activities. A corollary of this hypothesis is that motor neurons involved in many behavioral functions should branch in more neuropils than those active in fewer behaviors. In crayfish, the abdominal fast-flexor muscles are active only during the generation of the powerstroke for tailflips, whereas the slow-flexor muscles are involved in the maintenance of body posture. The slow flexors are thus active in many of the crayfish's behavioral activities. To test the generality of Altman's idea, we filled groups of crayfish fast-flexor and slow-flexor motor neurons with cobalt chloride and described their shapes with respect to the ganglionic structures through which they pass. Individual fast flexors were also filled intracellularly with HRP. Ganglia containing well-filled neurons were osmicated, embedded in plastic, and sectioned. Unstained sections were examined by light microscopy and pertinent sections were photographed. We found that the paths of the larger neurites were invariant, that the dendritic domains of fast and slow motor neurons occupied distinctive sets of neuropils, and that dendrites of slow motor neurons branched in more ganglionic structures than did those of fast motor neurons. These results are consistent with Altman's hypothesis.

NASA multipurpose airborne DIAL system and measurements of ozone and aerosol profiles.

An airborne differential absorption lidar (DIAL) system has been developed for the remote measurement of gas and aerosol profiles in the troposphere and lower stratosphere. The multipurpose DIAL system can operate from 280 to 1064 nm for measurements of ozone, sulfur dioxide, nitrogen dioxide, water vapor, temperature,pressure, and aerosol backscattering. The laser transmitter consists of two narrow linewidth Nd: YAG pumped dye lasers with automatic wavelength control. The DIAL wavelengths are transmitted with a 00-,usec temporal separation to reduce receiver system complexity. A coaxial receiver system is used to collect and optically separate the DIAL and aerosol lidar returns. Photomultiplier tubes detect the backscattered laser returns after optical filtering, and the analog signals from three tubes are digitized and stored on high-speed magnetic tape. Real-time gas concentration profiles or aerosol backscatter distributions are calculated and displayed for experiment control. Operational parameters for the airborne DIAL system are presented for measurements of ozone, water vapor, and aerosols in the 290-, 720-, and 600-nm wavelength regions, respectively. The first ozone profile measurements from an aircraft using the DIAL technique are discussed in this paper. Comparisons between DIAL and in situ ozone measurements show agreement to within +/-5 ppbv in the lower troposphere. Lidar aerosol data obtained simultaneously with DIAL ozone measurements are presented for a flight over Virginia and the Chesapeake Bay. DIAL system performance for profiling ozone in a tropopause folding experiment is evaluated, and the applications of the DIAL system to regional and global-scale tropospheric investigations are discussed.

Binding of thrombin to cultured human fibroblasts: evidence for receptor modulation.

Previous work from this laboratory has indicated that thrombin's influence on cell growth can be negative as well as positive. Addition of enzyme to actively growing or confluent cultures of human skin fibroblasts produced growth stimulation, whereas cultures receiving thrombin at the time of subculture displayed inhibited DNA synthesis and mitosis. The specific binding of [125I]thrombin to cells under stimulatory and inhibitory conditions has been studied. Fibroblasts receiving enzyme at subculture bound about two times more [125I]thrombin than those processed in the same way several hours later. The apparent dissociation constant for both groups was approximately 1.5 x 10(-8) M. In each case binding was saturable, although cells receiving enzyme at subculture showed a much higher rate of binding. Experiments were conducted in which enzyme was added to cells at various times after subculture. It was found that the ability of these fibroblasts to specifically bind [125I]thrombin decreased progressively over a 2-h period after subculture and then remained constant for at least 24 h. Evidence is also presented indicating that the binding of [125I]thrombin in both experimental groups was inversely dependent upon the culture density. The biological effects of elevated thrombin binding in cells receiving enzyme at subculture were examined. It was found that inhibited DNA synthesis and altered cellular morphology were directly to this parameter. This study suggests that fibroblasts may possess cryptic thrombin receptors that become exposed during subculture or after injury in vivo. These possibilities and the relationship of cell shape to the availability of thrombin receptors are discussed.

Differential effect of thrombin on the growth of human fibroblasts.

The ability of thrombin to alter the growth of human skin fibroblasts was studied under a variety of experimental conditions. In agreement with previous reports, we obtained a moderate level of cell growth in confluent cultures using 0.5-8.0 U/ml of thrombin. In subconfluent cultures, the effect was strikingly different and was found to be dependent upon the time in culture when the enzyme was added. Cultures exposed to thrombin 24 h after subculturing showed growth stimulation several days later. In contrast, thrombin added at the time of cell plating produced a complete block of DNA synthesis and cell growth that lasted for at least 3 d. Cells exposed to thrombin under these conditions were morphologically altered and smaller. These thrombin-induced effects were reversible and could be completely prevented by pretreatment of the enzyme with hirudin before it was added to the culture medium. Growth inhibition and altered morphology were found to be the result of changes generated in the growth medium by thrombin and could be blocked by higher serum concentrations. The results of this study indicate that thrombin's influence on cell growth can be stimulatory or inhibitory and suggest that the state of the cell surface determines the response.