Reversed-phase, high-pressure liquid chromatography of peptides and proteins with ion-pairing reagents.

Reversed-phase, high-pressure liquid chromatography has been successfully applied to the analysis of peptides and proteins by the addition of hydrophilic (for example, phosphoric acid) or hydrophobic (for example, hexanesulfonic acid) ion-pairing reagents, or both, to the mobile phase. Examples described included proteins such as insulin, glucagon, and 1-24 ACTH pentaacetate (ACTH is adrenocorticotrophic hormone).

Alcohol consumption patterns and attitudes toward drink-drive behaviours and road safety enforcement strategies.

BACKGROUND: Alcohol contributes to approximately 30% of all serious crashes. While the majority of drivers acknowledge the risks associated with drink-driving, a significant proportion of the population continue to engage in this behaviour. Attitudes towards drink-driving as well as personal alcohol consumption patterns are likely to underpin a driver's decision to drink-drive. These associations were explored in the current study. METHODS: A large (N=2994) cross-sectional online survey of a representative sample of drivers in Australia was conducted. Participants provided information about their own alcohol consumption patterns, drink-driving behaviour as well as attitudes towards drink-driving (own and others) and enforcement strategies. RESULTS: Alcohol consumption patterns differed according to age, gender and work status. Drivers who reported drink-driving behaviour and had high risk alcohol consumption patterns were less likely to agree that drink-driving leads to increased crash risk and more likely to agree they drink and drive when they believed they could get away with it. In contrast, drivers who did not report drink-driving and had low risk consumption patterns were more likely to report that the enforcement strategies are too lenient. Binary logistic regression showed that high risk alcohol consumption patterns and agreement from drivers that they drink and drive when they believe they can get away with it had the strongest associations with drink-driving. These findings highlight the relationships between one's drinking patterns, drink-drive behaviour and attitudes towards drink-driving and drink-driving enforcement CONCLUSIONS AND IMPLICATIONS: The patterns of associations that emerged suggest that drink-driving is the expression of a broader health issue for the most "at-risk" cohort of drinkers. The decision to drink and drive may result from a need borne from an alcohol dependent lifestyle exacerbated by a social acceptability of the behaviour and positive attitudes towards one's ability to drink-drive with few adverse consequences. Therefore, the broader alcohol consumption patterns of drink-drivers needs to be considered when targeting drink-drive reductions.

Mapping of proctolinlike immunoreactivity in the nervous systems of lobster and crayfish.

Whole-mount immunocytochemical techniques have been used to map candidate proctolin-containing cells in the central nervous systems of the lobster, Homarus americanus, and the crayfish, Procambarus clarkii. Proctolinlike immunoreactivity was detected in cell bodies and neuropil regions in all central ganglia, and immunoreactive axons were detected in most interganglionic connectives and nerve roots. Cell body staining was confined to fewer than 2% of all cells. Immunoreactive neurons include motoneurons, sensory neurons, neurosecretory cells, and interneurons. Colocalization of the proctolinlike antigen with other neurotransmitters was indicated in a number of cases. Many aspects of the distribution of immunoreactivity were similar in lobster and crayfish; however, staining differences were detected in a number of identified neurons and neural groups, including neurons that innervate the pericardial organs and hindgut motoneurons. Further studies of such neurons might provide interesting clues about the physiological functions of proctolin and the evolution of peptide transmission.

Neuropeptide proctolin (H-Arg-Try-Leu-Pro-Thr-OH): immunocytochemical mapping of neurons in the central nervous system of the cockroach.

Proctolinlike immunoreactivity was mapped in the central nervous system of the cockroach, Periplaneta americana, by using whole-ganglion immunoreacted preparations. The procedure for this immunohistochemical staining of whole-mounts is described. Immunoreactivity was confined to neuronal cell bodies and and processes. These were found in all ganglia of the CNS. The cells varied in the consistency and intensity of their staining. The occurrence and variability of staining is described in detail. Cell bodies were found in the dorsal, ventral, and lateral regions of the ganglia. The highest number of cell bodies was found in the terminal ganglion and the lowest number in the cerebral ganglion. Those in the cerebral ganglion occurred mainly in the tritocerebral lobes. The distribution of immunoreactive cell bodies correlated with results previously obtained by radioimmunoassay. Immunoreactive processes were detected in all interganglionic connectives and many ganglionic nerve roots. Dense ramifications of immunoreactive processes and variocosities were detected in many of the ganglia. The widespread presence of immunoreactivity suggests that proctolin has diverse central and peripheral functions. The mapping immunoreactive neuronal somata provides a valuable step in the identification of putative proctolin-containing neurons suitable for further biochemical, anatomical, and physiological analysis.

Nitrogen pollution: an assessment of its threat to amphibian survival.

The potential for nitrate to affect amphibian survival was evaluated by examining the areas in North America where concentrations of nitrate in water occur above amphibian toxicity thresholds. Nitrogen pollution from anthropogenic sources enters bodies of water through agricultural runoff or percolation associated with nitrogen fertilization, livestock, precipitation, and effluents from industrial and human wastes. Environmental concentrations of nitrate in watersheds throughout North America range from < 1 to > 100 mg/L. Of the 8,545 water quality samples collected from states and provinces bordering the Great Lakes, 19.8% contained nitrate concentrations exceeding those which can cause sublethal effects in amphibians. In the laboratory lethal and sublethal effects in amphibians are detected at nitrate concentrations between 2.5 and 100 mg/L. Furthermore, amphibian prey such as insects and predators of amphibians such as fish are also sensitive to these elevated levels of nitrate. From this we conclude that nitrate concentrations in some watersheds in North America are high enough to cause death and developmental anomalies in amphibians and impact other animals in aquatic ecosystems. In some situations, the use of vegetated buffer strips adjacent to water courses can reduce nitrogen contamination of surface waters. Ultimately, there is a need to reduce runoff, sewage effluent discharge, and the use of fertilizers, and to establish and enforce water quality guidelines for nitrate for the protection of aquatic organisms.

Impact of organochlorine contamination on levels of sex hormones and external morphology of common snapping turtles (Chelydra serpentina serpentina) in Ontario, Canada.

Recent research has suggested that contaminants in the environment may influence sex differentiation and reproductive endocrine function in wildlife. Concentrations of organochlorine contaminants (total polychlorinated biphenyls, pesticides) were higher in the blood plasma of snapping turtles from contaminated sites than in those from reference sites. The ratio of the precloacal length to the posterior lobe of the plastron (PPR) is sexually dimorphic in snapping turtles. There were significant reductions in the PPR at three contaminated sites versus two reference sites. The magnitude of the response was such that a significantly higher proportion of PPRs of males from a contaminated site (Cootes Paradise) overlapped with those of females than PPRs of males from a reference site (Lake Sasajewun). Observers can incorrectly identify the sex of turtles at the contaminated site based on secondary sexual characteristics alone. Unlike the changes to the morphology, there were few changes in 17 beta-estradiol or testosterone levels, and where differences occurred, there was more variation among reference sites than between the reference and contaminated sites. Our results suggest that environmental contaminants may affect sexually dimorphic morphology in snapping turtles without affecting circulating testosterone or estrogen levels in the adults.

A parallel evaluation of four automated hematology analyzers.

A parallel evaluation was performed on four automated hematology analyzers: the Celldyn 3000 (Unipath Corp., Mountain View, CA), the Coulter STKS (Coulter Electronics Inc., Hialeah, FL), the Sysmex NE-8000 (Baxter Healthcare Corp., McGaw Park, IL), and the Technicon H*2 (Miles Corp., Tarrytown, NY). The protocol included evaluation of the complete blood count and differential leukocyte count (DLC) parameters. The DLC evaluation was performed using the National Committee for Clinical Laboratory Studies H20-A protocol. Based on this evaluation, the authors could not identify a single instrument that was clearly superior to the others. Overall, the four instruments were found to be safe and effective for diagnostic use; however, there were areas in which their performance was less than optimal. Particular questions were raised regarding the clinical usefulness of instrumental "flags" to identify qualitative leukocyte abnormalities. The results are discussed in relation to the selection of instruments for specific clinical applications.

Flow-cytochemical differential leukocyte analysis with quantitation of neutrophil left shift. An evaluation of the Cobas-Helios analyzer.

The Cobas-Helios (Roche Diagnostic Systems, Inc., Branchburg, NJ) is a new, fully automated hematology analyzer that performs a complete blood count and differential leukocyte count (DLC), classifying leukocytes by flow-cytochemical technology. The DLC component of the Cobas-Helios was evaluated according to the National Committee for Clinical Laboratory Standards H20-A protocol. Instrument performance was acceptable with respect to all parameters investigated, including imprecision, inaccuracy and clinical sensitivity for the identification of quantitative and qualitative leukocyte abnormalities. In a minority of samples with neutrophil left shift, neutrophils tended to overlap the monocyte domain, resulting in overestimation of monocytes and underestimation of neutrophils. This problem did not affect clinical sensitivity and was generally associated with a positive instrumental left-shift flag. Flags for the identification of specific qualitative abnormalities of the leukocyte population (atypical lymphoid cells, nucleated red cells, blast cells, immature granulocytes and neutrophil left shift) performed well. In addition to a conventional five-part DLC, the Cobas-Helios also identifies and quantitates atypical lymphoid cells and "large immature cells," the latter corresponding to bands and immature granulocytes. Counts of atypical lymphoid cells and large immature cells correlated well with the equivalent cell classes as enumerated by the reference method of the National Committee for Clinical Laboratory Standards. The Cobas-Helios offers the most reliable quantitative index of neutrophil left shift currently available in a commercial automated DLC analyzer.

Correction of bone marrow nucleated cell counts for the presence of fat particles.

Although the use of bone marrow transplantation has increased greatly in recent years, the quality control procedures used in bone marrow processing laboratories remain less than ideal. Accurate marrow total nucleated cell (TNC) counts are essential for effective monitoring of bone marrow collection and processing. Aspirated marrow is variably contaminated by fat particles, resulting in overestimation of marrow TNC by automated analyzers. A recently-marketed hematological analyzer (Cobas-Helios; Roche Diagnostic Systems, Branchburg, NJ) offers the potential to correct marrow TNC counts for fat particles using available software. The authors investigated the accuracy of corrected TNC counts on 21 marrow samples, using a visual chamber count as the reference method. The correction methods studied were software correction, using the Cobas-Helios differential system, and replacement of the sample plasma with saline. Uncorrected automated marrow TNC counts (mean, 28.4 x 10(9)/L) were significantly higher than the visual reference counts (mean, 23.1 x 10(9)/L). Neither the mean corrected automated count (24.3 x 10(9)/L) nor the mean saline replaced count (24.6 x 10(9)/L) differed significantly from the mean visual reference count. For both the corrected automated and saline replaced counts, 20 of the 21 data points (95%) fell within a 95% confidence interval computed for the reference method. The authors conclude that both the corrected automated method, using the Cobas-Helios, and the saline replacement method are acceptable alternatives to the visual chamber count.

Health of tree swallows (Tachycineta bicolor) nesting in pesticide-sprayed apple orchards in Ontario, Canada. I. Immunological parameters.

The degree of pesticide exposure and its effects on the immune system and its development were determined in 16-d-old tree swallow (Tachycineta bicolor) chicks from 4 sprayed apple orchards and three nonsprayed sites in southern Ontario, Canada, during 1994-1995. Persistent contaminant residues were measured in tree swallow eggs and in each chick hepatic ethoxyresorufin O-deethylase (EROD) activity; body, immune organ, and liver masses; lymphocyte blastogenesis response; respiratory burst and phagocytic responses; hemarological evaluation; and histological development of thymus, bursa of fabricius, and spleen were determined. Chemicals sprayed on apple orchards were mainly ethylene bisdithiocarbamate and myclobutanil fungicides and organophosphorus, carbamate, and synthetic pyrethroid insecticides. During the period between oviposition of the first egg in each nest to d 16 after hatching, individual nests in orchards were exposed to between 4 and 11 individual chemical applications and up to 3 mixtures of pesticide sprays. Concentrations of pesticides, polychlorinated biphenyls (PCBs), and lead and arsenic residues in tree swallow eggs and liver were low and not variable among sites except p,p'-DDE, which was as high as 2.29 microg/g wet weight in eggs. EROD activity was not different among sites. Organochlorine and trace metal residues and EROD activity were not correlated with any immune parameter. In sprayed birds, we found a significantly increased blastogenic response to pokeweed mitogen (12.5 microg/ml). However, nests were initiated over a period of several weeks and we also found changes in other tree swallow immune parameters that were related to the date of chick collection. Hematological parameters, bursal and thymic masses, phagocytic response, and thymic development were all correlated with the day the chicks were 16 d of age. After accounting for the collection date of birds from each nest, we found cell proliferation in the cortex and delayed thymic involution correlated positively with increasing spray exposure. We also found that birds in sprayed orchards were slightly anemic compared to birds from nonsprayed sites, and there were smaller bursal masses and an increase in relative heterophil concentrations in the sprayed orchard birds. The local inflammation may have been caused by trematode parasite infections, although pesticide exposure also correlated positively with these parameters. This is the first study of the immunology and effects of current pesticide exposures in wild passerines; therefore it is difficult to predict the long-term consequences of the apparent stimulated immune systems in sprayed birds. However, some environmental contaminants that overtly stimulate the immune system in mammals have induced hypersensitivity and/or autoimmunity. Therefore we speculate that these effects are possible in tree swallows.

Health of tree swallows (Tachycineta bicolor) nesting in pesticide-sprayed apple orchards in Ontario, Canada. II. Sex and thyroid hormone concentrations and testes development.

To investigate the effects of pesticides on wild birds, sex (17beta-estradiol; testosterone) and thyroid (triiodothyronine (T3) hormone concentrations, body mass, and testes mass were measured and the development of testes was evaluated in wild tree swallows (Tachycineta bicolor) nesting in four sprayed apple orchards and three nonsprayed sites in southern Ontario, Canada, in 1995-1996. In orchards, birds were exposed to asmany as 11 individual spray events and five sprays of mixtures of chemicals. Residues of organochlorine pesticides, PCBs, lead, and arsenic concentrations were low and not variable among sites except p,p'-DDE concentrations, which ranged from 0.36 to 2.23 microg/g wet weight in eggs. These persistent compounds were not correlated with any endocrine response measured in tree swallows. In 16-d-old male tree swallow chicks, body mass and concentrations of 17beta-estradiol (estradiol), testosterone, and T3 in plasma showed no significant differences between sprayed and nonsprayed groups and among sites within those groups. However, T3 concentrations were slightly elevated in the sprayed group compared to the nonsprayed group, and there was a significant and positive correlation between T3 and the number of mixtures of sprays applied during egg incubation through chick rearing. In 16-d-old female chicks, there were no significant differences among spray treatments or sites and no correlations with spray exposure for testosterone, estradiol, or T3 in plasma. Body mass was correlated positively with T3 and negatively with estradiol but showed no differences among spray exposure groups or sites. Histology of testes of 16-d-old male chicks indicated there were no significant differences among sprayed and nonsprayed birds in testes mass, area, or diameter, or the presence of Leydig cells in the interstitium, the distribution of the Sertoli cells, or the occurrence of heterophils in the testicular interstitium. For the percentage of spermatogonia present on the basement membrane, there were significant differences among sites, but these differences were not specifically associated with spray exposure. However, there was a marginally significant trend between increasing occurrence of a disrupted Sertoli cell population on the seminiferous tubular basement membranes as the number of mixtures of pesticides sprayed during chick rearing increased. In adult male and female parent tree swallows, there were no differences in hormone concentrations between birds from sprayed and nonsprayed sites. Nor were there any significant correlations between the concentration of any hormone and collection date, body mass, or any type of spray exposure for adults. The correlations between increasing pesticide exposure and abnormal thyroid hormone and testes development in male chicks indicate that further reductions of pesticide use in orchards may benefit the health of birds that nest there. However, it is unclear which of these pesticides or spray mixtures are responsible for these effects, and this needs to be examined in future studies.

Separation of the apoprotein components of human very low density lipoproteins by ion-paired, reversed-phase high performance liquid chromatography.

A number of crude apolipoprotein samples isolated from human very low density lipoproteins (VLDL) were analyzed by reversed phase high performance liquid chromatography. The mobile phase consisted of a 1% solution of the polar ion-pairing reagent triethylammonium phosphate. A slow, nonlinear gradient of acetonitrile (37--42%) was used to elute the apolipoproteins. The order of elution was as follows: apolipoprotein CX, apolipoprotein C-I, apolipoprotein C-III2, apolipoprotein C-III1, apolipoprotein C-IIIQ and apolipoprotein C-II. This order is consistent with the known polarity of the proteins, i.e., the most nonpolar, apolipoprotein C-II, was the last to be eluted, whereas apolipoprotein C-I, with the lowest nonpolar surface area eluted first. The recovery of the individual apolipoproteins was 80--95% and the individual peaks were characterized by amino acid analysis, UV absorption spectra amd chromatography of pure protein standards.

Environmental contamination and developmental abnormalities in eggs and hatchlings of the common snapping turtle (Chelydra serpentina serpentina) from the Great Lakes-St Lawrence River basin (1989-1991).

During 1989-1991, we assessed developmental abnormalities in embryos and hatchlings from eggs of the common snapping turtle (Chelydra serpentina serpentina). Eggs were collected and artificially incubated from eight sites in Ontario, Canada and Akwesasne/New York, USA. In eggs from the same clutches we measured 20 organochlorine pesticides, 48 polychlorinated biphenyl (PCBs) congeners including 6 non-ortho PCBs, 8 polychlorinated dibenzodioxins (PCDDs), 14 polychlorinated dibenzofurans (PCDFs) and total mercury. We found a significant increase in abnormal development with increasing polychlorinated aromatic hydrocarbon exposure in eggs, particularly PCDD and PCDF concentrations. In contrast, the risk of abnormality was not significantly higher as toxic equivalent concentrations increased in eggs. We also found significant 7-ethoxyresorufin O-deethylase and Cytochrome P4501A responses in livers of hatchling turtles from Lake Ontario relative to hatchlings from a clean, inland site whereas we did not find any evidence of porphyria in the hatchlings from either site.

Polychlorinated biphenyls and organochlorine pesticides in plasma and the embryonic development in Lake Erie water snakes (Nerodia sipedon insularum) from Pelee Island, Ontario, Canada (1999).

From three locations along a 34-km shoreline of Pelee Island, Ontario, 30 gravid female Lake Erie water snakes (Nerodia sipedon insularum) were sampled to determine the organochlorine (OC) contaminant levels in plasma and the number of live and dead embryos present in the body cavity. Plasma was analyzed for 59 polychlorinated biphenyl (PCB) congeners and 14 organochlorine pesticides. Concentrations of pesticides were low (< or =0.1 ng/g wet wt) in all snakes, but there was significant variation in mean PCB concentrations in plasma from among the sampling locations on Pelee Island. Snakes (n = 5) from the West shore and dock area of the island had significantly higher PCB concentrations (90.4 +/- 15.0 ng/g wet wt) in plasma than those from Lighthouse Point (n = 5; 34.4 +/- 13 ng/g wet wt) and the south shore of the island (n = 5; 29.4 +/- 16.3 ng/g wet wt). Body mass of the female snakes ranged from 252 to 880 g, and mean masses were not significantly different among sample sites. The number of live embryos found ranged from 13 to 46 female snakes and no dead embryos were detected. There were significant positive correlations among body mass, snout-vent length, and number of young per female. There were no significant correlations among body mass, snout-vent length, number of young per female, or per-gram body mass of female snakes and contaminant concentrations in plasma. It was concluded that an interim estimate of a no-effect level on embryonic survival in N. sipedon insularum may be a maximum average concentration of 90.4 ng/g wet wt PCBs and a maximum average concentration of 3.6 ng/g wet wt p,p'-dichloro-diphenyl-dichloroethylene in plasma.

Effects of azinphos-methyl on cholinergic responses and general health in zebra finches (Taeniopygia guttata) after previous treatment with p,p'-DDE.

Although organochlorine (OC) pesticides were replaced with organophosphates (OPs) in the early 1970s, they continue to persist in orchard environments today. Extensive research has been conducted to determine the effects of currently used OPs on cholinesterase (ChE) activity; however, although OCs continue to be prevalent in areas of previous use, few studies have looked at the toxicity of a combination of residual OC compounds with currently used OP pesticides. The focus of our study was to determine the effects of azinphos-methyl (a common OP used in apple orchards today) on ChE activity and general health in zebra finches (Taeniopygia guttata) previously exposed to p,p'-DDE (a commonly detected metabolite of DDT). The main results of our study were as follows: (1) azinphos-methyl alone caused a dose-dependent inhibition of plasma and brain ChE activity; (2) p,p'-DDE in combination with azinphos-methyl did not change azinphos-methyl inhibition of ChE activity; and (3) there were suggestions of immunostimulation in birds dosed 1 year previously to p,p'-DDE and of anemia when p,p'-DDE was combined with azinphos-methyl; however, there was no dose-response for these parameters in birds subsequently dosed with p,p'-DDE.

Serotonin immunoreactive neurons in the central nervous system of an insect (Periplaneta americana).

Serotonin-like immunoreactivity was mapped in the central nervous system (CNS) of the cockroach, Periplaneta americana. Immunoreactive staining occurred in every ganglion of the CNS. The largest numbers of immunoreactive somata were detected in the optic lobes and the brain, and lowest numbers in the first and second thoracic ganglia. Dense stained fibers, ramifications, and varicosities were found in all ganglia, and numerous axon like processes occurred in all interganglionic connectives. Immunoreactive processes were not, however, detected in most of the peripherally projecting nerve roots. Processes were found only in roots of the suboesophageal ganglion and the tritocerebral lobes of the brain. A comparison of the map for serotonin immunoreactivity with one generated for the pentapeptide transmitter proctolin suggests that the two systems overlap only in the suboesophageal ganglion and the tritocerebrum. The amine and peptide may co-occur in neurons in these regions. The serotonin immunoreactive system appeared significantly different from the octopaminergic system of the ventral nerve cord. Seventy-two potentially identifiable immunoreactive cells were located in the cockroach CNS. Some of these may be suitable for physiological study of the functional role of serotonin.

Chlorinated hydrocarbon concentrations in plasma of the Lake Erie water snake (Nerodia sipedon insularum) and northern water snake (Nerodia sipedon sipedon) from the Great Lakes basin in 1998.

From the Great Lakes basin, concentrations of 59 congener-specific polychlorinated biphenyls (PCBs) and 14 organochlorine pesticides were measured in blood plasma of northern water snake (Nerodia sipedon sipedon) and Lake Erie water snake (Nerodia sipedon insularum), which is endangered in Canada. In 1998, four male adult Lake Erie water snakes were sampled from Pelee Island, western Lake Erie; four male northern water snakes were sampled at Little Lake, about 20 km north of Parry Sound in central Ontario; and four adult gravid female northern water snakes were sampled from Garden Island, eastern Lake Ontario. The blood plasma was pooled by site for a total of three samples analyzed. The Pelee Island sample from male Lake Erie water snakes contained less than half the lipid concentration (0.349%) than samples from the other sites, but it was the most contaminated with PCBs, even on a wet weight basis. Summed concentration of individual PCBs in the Pelee Island sample was 167 ng/g (wet weight), which was 14-fold higher than the next most contaminated sample, which was from Little Lake. The plasma sample from Little Lake contained 12 ng/g (WW) and was four times more contaminated with PCBs than the sample from female snakes from Garden Island, Lake Ontario. Organochlorine pesticide concentrations in plasma were relatively similar among sites. None of the pesticides was found above trace concentrations (0.1-0.9 ng/g) except pp'-DDE, which occurred at 2-5 ng/g among sites. PCB congener patterns in the Lake Erie water snakes were compared to PCB patterns in plasma of common snapping turtle (Chelydra serpentina serpentina) from Lake Ontario, herring gull eggs (Larus argentatus) from western Lake Erie, and mudpuppy eggs (Necturus maculosus) from the Detroit River. The PCB patterns in water snake and herring gull sample were most similar, followed by the pattern in snapping turtle plasma. The presence of more lower-chlorinated chlorobiphenyls in the mudpuppy eggs relative to the other species made this sample distinct from the water snake, gull, and turtle.

Neuropeptide proctolin associated with an identified skeletal motoneuron.

Our objective was to find large and identifiable peptide-containing neurons and their postsynaptic targets. For this, a whole mount immunohistochemical method was used to locate cell bodies of neurons in the cockroach central nervous system immunoreactive to antibodies raised against the pentapeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH). The morphology and projections of the immunoreactive neurons were investigated by combining intracellular dye injection and electrophysiological and immunochemical procedures. The presence of proctolin in specific immunoreactive neurons was check by high pressure liquid chromatography combined with bioassay applied to extracts made from individually identified and isolated cell bodies. Using these approaches, we have identified a large proctolin-containing motoneuron which innervates muscles in a proximal segment of the cockroach leg. This study establishes a well characterized cellular preparation in which the physiological actions and functions of a peptide-containing neuron can be studied in vivo.

Vertical motion detectors and their synaptic relations in the third optic lobe of the fly.

The synaptic relations of the giant vertical cells in the lobula plate of the fly were investigated using electron microscopical procedures and Lucifer yellow dye backfill and injection techniques. Histological features of the giant vertical cells are described. The giant vertical cells are exclusively postsynaptic in the lobula plate. They function to integrate input from dense arrays of chemical synapses and have a wide spatial input from the lobula plate. The giant vertical cells are postsynaptic to perpendicularly occurring cells. There are two classes of cells presynaptic to the vertical cells, one of which contains large dense-core vesicles. The giant vertical cells are not the only cells postsynaptic to these two classes of perpendicular cells. A second group of smaller tangential cells, the twin vertical cells, were also found postsynaptic to many of the same cells that synapsed with the giant vertical cells. The twin vertical cells and the giant vertical cells are therefore integrating some of the same information in the lobula plate.